Method for synthesizing polynucleotides

ABSTRACT

The present invention realized isothermal and rapid polynucleotide synthesis by using as templates polynucleotides having a structure capable of forming loops, and combining a plurality of primers capable of providing a starting point for complementary strand synthesis to such loops. If the LAMP method is applied, all reactions can be carried out isothermally and rapidly since the template polynucleotides themselves can also be synthesized by an isothermal reaction.

This application is a national stage application under 35 USC 371 ofPCT/JP01/08142 filed Sep. 19, 2001, which claims the priority benefit ofJapanese Application No. 2000-283862 filed Sep. 19, 2000.

TECHNICAL FIELD

The present invention relates to a method for synthesizingpolynucleotides.

BACKGROUND ART

Template-dependent nucleic acid synthesis methods using the PolymeraseChain Reaction (PCR) method have served as a major driving force forresearch in bioscience fields in recent years. The PCR method has madeit possible to exponentially amplify nucleic acids composed of anucleotide sequence complementary to a template by using a small amountof double-stranded nucleic acid as template. PCR is currently widelyused as a tool for gene cloning and detection. In the PCR method, oneset of primers comprising nucleotide sequences complementary to bothends of a target nucleotide sequence is used. One of the primers isdesigned to anneal to the elongation product generated by the otherprimer. In this manner, a synthesis reaction progresses in whichannealing to a mutual elongation product and complementary strandsynthesis are repeated, enabling exponential amplification to beachieved.

In the PCR method, complex temperature control is essential. A specialreaction apparatus must be used to accommodate this complex temperaturecontrol. Thus, it is difficult to perform PCR at hospital bedsides oroutdoors. In addition, improvement of reaction specificity has been animportant problem for known complementary strand synthesis reactions.For example, in the PCR method, when a complementary strand synthesisproduct is used as a new template, the region to which the primeranneals is not a nucleotide sequence derived from the sample in thestrict sense, but rather is merely a copy of the nucleotide sequence ofthe primer. Thus, it is typically difficult to recognize slightdifferences in nucleotide sequences using a PCR primer.

As one method for solving these problems, the LAMP method was invented(Loop-mediated isothermal amplification) (Nucleic Acid Res. 2000, Vol.28, No. 12 e63, WO 00/28082). The LAMP method makes it possible toanneal to a template polynucleotide its own 3′-end to serve as astarting point for complementary strand synthesis, while also enablingan isothermal complementary strand synthesis reaction by combining aprimer that is annealed to the loop formed at this time. In addition, inthe LAMP method, since the 3′-end anneals to a region derived from thesample, a nucleotide sequence checking mechanism functions repeatedly.As a result, it has become possible to identify even slight differencesin nucleotide sequences.

When detecting a target nucleotide sequence based on a complementarystrand synthesis reaction such as LAMP or PCR, there is a closerelationship between the time required for the reaction and detectionsensitivity. In other words, allowing the reaction to proceed for aslong a time as possible until the reaction reaches a plateau is acondition for achieving high detection sensitivity. In the case of knowncomplementary strand synthesis reactions like LAMP and PCR, the reactionreaches a plateau in about 1 hour. In other words, it may be said that areaction time of about 1 hour is required in order to obtain maximumsensitivity. Although a reaction time of 1 hour is not that long, itwould be even more useful if an even shorter reaction time can berealized without sacrificing detection sensitivity or procedural ease.

Following the identification of the genome draft, science is entering apost-genome era. There is a growing need for analyzing single nucleotidepolymorphisms (SNPs) and gene function as well as gene diagnosis basedon the results of those analyses. Thus, the development of a technologythat enables gene nucleotide sequences to be analyzed more accuratelyand rapidly is becoming an important issue not only in terms of rapidlycarrying out functional analysis, but also with respect to practicalapplication of the results of gene function analysis in actual clinicalsettings.

DISCLOSURE OF THE INVENTION

The objective of the present invention is to provide a method that canrapidly carry out a complementary strand synthesis reaction using apolynucleotide as template.

The inventor conducted extensive research on reaction conditions forcomplementary strand synthesis to solve the above problems. As a result,it was found that the combination of primers used for complementarystrand synthesis and the 3′-end serving as the starting point forcomplementary strand synthesis was intimately related to the reactionrate of complementary strand synthesis. Moreover, it was revealed thatthe reaction efficiency of complementary strand synthesis can beimproved by combining a template polynucleotide having a specificstructure with a primer capable of providing the starting point forcomplementary strand synthesis at a specific location of thispolynucleotide, thereby leading to the completion of the presentinvention. Namely, the present invention relates to the followingpolynucleotide synthesis method and the application thereof.

[1] A method for synthesizing a polynucleotide comprising the steps ofmixing the following elements 1 to 5, and incubating under conditionsthat allow template-dependent complementary strand synthesis using theDNA polymerase in 4:

-   1: a template polynucleotide that:

(a) has a target nucleotide sequence comprising at least one set ofcomplementary nucleotide sequences,

(b) forms a loop capable of base pairing when the complementarynucleotide sequence of (a) hybridizes,

(c) forms a loop by the annealing of its 3′-end to itself, and

(d) whose 3′-end annealed to itself can be a starting point forcomplementary strand synthesis using itself as template;

-   2: at least two types of primers providing starting points for    complementary strand synthesis at different locations on the    template polynucleotide loop;-   3: at least one type of primer providing a starting point for    complementary strand synthesis at a location different from the    primers of 2 in a loop formed by template polynucleotide and/or an    elongation product produced by the annealing of the primers of 2 to    the template polynucleotide;-   4: a DNA polymerase catalyzing complementary strand synthesis    accompanying strand displacement; and,-   5: a substrate for complementary strand synthesis.

[2] The method of [1], wherein said template polynucleotide has on its5′-end a nucleotide sequence complementary to an arbitrary region of itsown nucleotide sequence.

[3] The method of [2], wherein said template polynucleotide is producedby the following steps of:

a) annealing a first primer to a target nucleotide sequence, andconducting a complementary strand synthesis. reaction using this as astarting point, wherein said first primer (i) can provide at the 3′-enda starting point for complementary strand synthesis to a region thatdefines the 3′-side of one of the strands that compose the targetnucleotide sequence, and (ii) has on its 5′-side a nucleotide sequencecomplementary to an arbitrary region of a complementary strand synthesisreaction product that uses this primer as a starting point;

b) placing, in a condition that allows base pairing, the region to wherea second primer is to anneal in the elongation product of the firstprimer synthesized in step a), wherein said second primer (i) has on its3′-end a nucleotide sequence providing a starting point forcomplementary strand synthesis to a region that defines the 3′-side of atarget nucleotide sequence in the elongation product that uses the firstprimer as a starting point, and (ii) has on its 5′-side a nucleotidesequence that is complementary to an arbitrary region of a complementarystrand synthesis reaction product that uses this primer as an startingpoint;

c) annealing said second primer to the region that can form base pairingin step b), and carrying out complementary strand synthesis using thisas a starting point; and,

d) annealing the 3′-end of the elongation product of the second primersynthesized in step c) to itself, and carrying out complementary strandsynthesis using itself as template.

[4] The method of [3], wherein the two types of primers are a firstprimer and a second primer, and at least one type of the primers is aloop primer providing, between a region derived from each primer in theelongation product of the first primer or second primer and thearbitrary region with respect to each primer, a starting point forcomplementary strand synthesis.

[5] The method of [4], wherein said loop primer is (i) a first loopprimer providing, between a region derived from the first primer in theelongation product of the first primer and the arbitrary region withrespect to the first primer, a starting point for complementary strandsynthesis, and (ii) a second loop primer providing, between a regionderived from the second primer in the elongation product of the secondprimer and the arbitrary region with respect to the second primer, astarting point for complementary strand synthesis.

[6] The method of [4], wherein said loop primer further comprises on its5′-end a nucleotide sequence complementary to the arbitrary region.

[7] The method of [3], wherein each product of the first primer orsecond primer is converted to a single strand by displacing theelongation product of the first primer and/or second primer according tocomplementary strand synthesis from an outer primer that provides astarting point for complementary strand synthesis to the 3′-side of atemplate with respect to the first primer or second primer in step b)and/or step c).

[8] The method of [3], wherein the target nucleotide sequence is presentas a double-stranded polynucleotide in step a), and the region to whichthe first primer is annealed is made to form base pair bonds accordingto a complementary strand synthesis reaction using the arbitrary primeras a starting point.

[9] The method of [8], wherein step a) is carried out in the presence ofa melting temperature regulator.

[10] The method of [9], wherein the melting temperature regulator is atleast one compound selected from the group consisting of betaine,proline, dimethylsulfoxide, and trimethylamine N-oxide.

[11] A method for amplifying a template polynucleotide, comprising thestep of repeating complementary strand synthesis using the templatepolynucleotide as template according to the method of [1] or [5], andalso carrying out another polynucleotide synthesis reaction according tothe method of [1] or [5] using the elongation product resulting from thesynthesis reaction as a new template polynucleotide.

[12] A method for detecting a target nucleotide sequence in a sample,comprising the step of carrying out the amplification method of [11],and correlating the production of the amplification reaction productwith the presence of a target nucleotide sequence.

[13 ] The method of [12], wherein the method of [11] is carried out inthe presence of a polynucleotide detecting agent, and whether or not anamplification reaction product is produced is observed based on a signalchange of the detecting agent.

[14] A method for detecting a mutation in a target nucleotide sequenceaccording to the detection method of [12], the method comprising thesteps of (i) blocking at least one of the complementary strand synthesisreactions selected from the complementary strand synthesis reactionscomposing the amplification method, when the target nucleotide sequenceis not the predicted nucleotide sequence, and (ii) observing theinhibition of the amplification reaction.

[15] The method of [14] that uses the following first primer and secondprimer, wherein at least either the first primer or second primercomprises a checking sequence on its 5′-side,

wherein, a checking sequence refers to a nucleotide sequence in which(i) when a nucleotide sequence that composes a specific region is notthe predicted nucleotide sequence, a mismatch occurs at the time whenthe 3′-end of the complementary strand synthesized using the checkingsequence as template anneals to the target nucleotide sequence, or itscomplementary strand, and (ii) a complementary strand synthesis reactionthat starts by using the 3′-end as a starting point is inhibited by thismismatch,

the first primer (i) can provide at its 3′-end a starting point forcomplementary strand synthesis to a region that defines the 3′-side ofone of the strands that composes a target nucleotide sequence, and (ii)has on its 5′-side a nucleotide sequence that is complementary to thearbitrary region of the complementary strand synthesis reaction productthat uses this primer as a starting point, and

the second primer (i) has a nucleotide sequence on its 3′-end thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of a target nucleotide sequence in anelongation product that uses the first primer as a starting point, and(ii) has on its 5′-side a nucleotide sequence that is complementary tothe arbitrary region of the complementary strand synthesis reactionproduct that uses this primer as a starting point.

[16] The method of [15], wherein when the nucleotide sequence thatcomposes the specific region is not the predicted nucleotide sequence, amismatch occurs in the 2nd to 4th nucleotides from the 3′-end of thecomplementary strand at the time when the complementary strandsynthesized by using a checking sequence as template anneals to thetarget nucleotide sequence, or its complementary strand.

[17] The method of [15] that uses the following first loop primer and/orsecond loop primer as loop primers; provided that, when the loop primercomprises on its 5′-side a nucleotide that is complementary to thearbitrary region arranged on the 5′-side of the primer, or when thenucleotide sequence arranged on the 5′-side of the primer is a checkingsequence, a sequence in which the nucleotide for providing the mismatchin the checking sequence differs from the checking sequence is arrangedon the 5′-side of the loop primer:

first loop primer: provides, between a region derived from a firstprimer in an elongation product of the first primer and the arbitraryregion with respect to the first primer, a starting point forcomplementary strand synthesis;

second loop primer: provides, between a region derived from a secondprimer in an elongation product of the second primer and the arbitraryregion with respect to the second primer, a starting point forcomplementary strand synthesis.

[18] The method of [17], wherein, when the nucleotide sequence thatcomposes the specific region is not the predicted nucleotide sequence, amismatch occurs in the 2nd to 4th nucleotides from the 3′-end of thecomplementary strand at the time when the complementary strandsynthesized using a checking sequence as a template anneals to thetarget nucleotide sequence, or its complementary strand, and wherein,the nucleotide sequence arranged on the 5′-side of the first loop primerand/or second loop primer differs in the nucleotide that causes themismatch in the checking sequence.

[19] A kit for amplifying a target nucleotide sequence comprising:

a) a first primer that (i) can provide at its 3′-end a starting pointfor complementary strand synthesis to a region that defines the 3′-sideof one of the strands that compose the target nucleotide sequence, and(ii) has on its 5′-side a nucleotide sequence that is complementary tothe arbitrary region of a complementary strand synthesis reactionproduct that uses this primer as an starting point;

b) a second primer that has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of a target nucleotide sequence in anelongation product that uses the first primer as an starting point, and(ii) on its 5′-side a nucleotide sequence that is complementary to thearbitrary region of a complementary strand synthesis reaction productthat uses this primer as a starting point;

c) a first loop primer that provides, between a region derived from thefirst primer in an elongation product of the first primer and thearbitrary region with respect to the first primer, a starting point forcomplementary strand synthesis;

d) a second loop primer that provides, between a region derived from thesecond primer in an elongation product of the second primer and thearbitrary region with respect to the second primer, a starting point forcomplementary strand synthesis;

e) a DNA polymerase catalyzing complementary strand synthesisaccompanying strand displacement; and,

f) a substrate for complementary strand synthesis.

[20] The kit of [19] that further comprises:

g) an outer primer that can provide a starting point for complementarystrand synthesis to the 3′-side of a template of the first primer and/orsecond primer.

[21] The kit of [19], wherein the first primer and/or second primercomprise a checking sequence on the 5′-side.

[22] The kit of [19] comprising the following first loop primer and/orsecond loop primer as a loop primer; provided that, when the loop primercomprises on its 5′-side a nucleotide sequence that is complementary tothe arbitrary region arranged on the 5′-side of the primer, or when thenucleotide sequence arranged on the 5′-side of the primer is a checkingsequence, the nucleotide for providing the mismatch in the checkingsequence arranges a sequence that differs from the checking sequence onthe 5′-side of the loop primer:

first loop primer: provides, between a region derived from the firstprimer in an elongation product of the first primer and the arbitraryregion with respect to the first primer, a starting point forcomplementary strand synthesis; and,

second loop primer: provides, between a region derived from the secondprimer in an elongation product of the second primer, and the arbitraryregion with respect to the second primer, a starting point forcomplementary strand synthesis.

[23] A method for amplifying a polynucleotide comprising the steps ofmixing the following elements a) through g), and incubating underconditions that enable a complementary strand synthesis reactionaccompanying strand displacement:

a) a first primer that (i) can provide at its. 3′-end a starting pointfor complementary strand synthesis to a region that defines the 3′-sideof one of the strands that compose a target nucleotide sequence, and(ii) has on its 5′-side a nucleotide sequence that is complementary tothe arbitrary region of a complementary strand synthesis reactionproduct that uses this primer as a starting point;

b) a second primer that has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of a target nucleotide sequence in anelongation product that uses the first primer as a starting point, and(ii) on its 5′-side a nucleotide sequence that is complementary to thearbitrary region of a complementary strand synthesis reaction productthat uses this primer as a starting point;

c) a first loop primer that can provide, between a region derived fromthe first primer in an elongation product of the first primer and thearbitrary region with respect to the first primer, a starting point forcomplementary strand synthesis;

d) a second loop primer that can provide, between a region derived fromthe second primer in an elongation product of the second primer and thearbitrary region with respect to the second primer, a starting point forcomplementary strand synthesis;

e) a DNA polymerase catalyzing complementary strand synthesisaccompanying strand displacement;

f) a substrate for complementary strand synthesis; and,

g) a test polynucleotide comprising a target nucleotide sequence.

[24] The method of [23] that further comprises:

h) an outer primer that provides a starting point for complementarystrand synthesis to the 3′-side of a template with respect to the regionin which the 3′-end of the first primer and/or second primer anneals tothe target nucleotide sequence.

[25] A method for determining whether a specific nucleotide in a targetnucleotide sequence is the first nucleotide or the second nucleotide,comprising the step of mixing the following elements a) through d), andincubating under conditions that enable a complementary strand synthesisreaction accompanying strand displacement, wherein formation rate and/orformed amount of the amplification product is measured by any one of theprimer sets in a) selected from the group consisting of:

a)

-   (1): first nucleotide inner primer pair and first nucleotide loop    primer pair-   (2): first nucleotide inner primer pair and second nucleotide loop    primer pair-   (3): second nucleotide inner primer pair and first nucleotide loop    primer pair, and-   (4): second nucleotide inner primer pair and second nucleotide loop    primer pair;

wherein, the first nucleotide inner primer pair and the secondnucleotide inner primer pair are both primer pairs consisting of thenext first inner primer and second inner primer, and in the firstnucleotide primer pair, a complementary strand synthesis reaction usingas the starting point the 3′-end of the complementary strand synthesizedusing the 5′-sides of the first inner primer and second inner primer asa template is not inhibited when the specific nucleotide in the targetnucleotide sequence is the first nucleotide, but is inhibited when it isthe second nucleotide;

in the second nucleotide inner primer pair, a complementary strandsynthesis using as the starting point the 3′-end of a complementarystrand synthesized using the 5′-sides of the first inner primer andsecond inner primer as a template is not inhibited when the specificnucleotide in the target nucleotide sequence is the second nucleotide,but is inhibited when it is the first nucleotide;

the first inner primer has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of one of the strands that compose a targetnucleotide sequence, and (ii) on the 5′-side a nucleotide sequence thatis complementary to the arbitrary, region of a complementary strandsynthesis reaction product that uses this inner primer as an startingpoint;

the second inner primer has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of a target nucleotide sequence in anelongation product that uses the first inner primer as an startingpoint, and (ii) on the 5′-side a nucleotide sequence that iscomplementary to the arbitrary region of a complementary strandsynthesis reaction product that uses this inner primer as an startingpoint;

the first nucleotide loop primer pair and the second nucleotide loopprimer pair are both pairs consisting of the next first loop primer andsecond loop primer, and in the first nucleotide loop primer pair, acomplementary strand synthesis reaction using as the starting point the3′-end of the complementary strand synthesized using the 5′-sides of thefirst loop primer and second loop primer as template is not inhibitedwhen the specific nucleotide in the target nucleotide sequence is thefirst nucleotide, but is inhibited when it is the second nucleotide;

in the second nucleotide inner primer pair, a complementary strandsynthesis reaction using as the starting point the 3′-end of acomplementary strand synthesized using the 5′-sides of the first loopprimer and second loop primer as a template is not inhibited when thespecific nucleotide in the target nucleotide sequence is the secondnucleotide, but is inhibited when it is the first nucleotide;

the first loop primer provides, between a region derived from the firstinner primer in an elongation product of the first inner primer and thearbitrary region with respect to the first inner primer, a startingpoint for complementary strand synthesis, and

the second loop primer provides, between a region derived from thesecond inner primer in an elongation product of the second inner primerand the arbitrary region with respect to the second inner primer, astarting point for complementary strand synthesis;

b) a DNA polymerase catalyzing complementary strand synthesisaccompanying strand displacement;

c) a substrate for complementary strand synthesis; and

d) a test polynucleotide comprising a target nucleotide sequence.

[26] The method of [25] that further comprises:

e) an outer primer that provides a staring point for complementarystrand synthesis to the 3′-side of a template with respect to the regionin which the 3′-end of the first primer and/or second primer anneals tothe target nucleotide sequence.

The polynucleotides used in the present invention may be DNA, RNA, orchimeric molecules thereof. The polynucleotides may be natural nucleicacids, as well as artificially synthesized nucleic acids. Further,nucleotide derivatives having partially or completely artificialstructures are also included in the polynucleotides of the presentinvention, so long as they can form base pairs and if necessary can beused as a template in complementary strand synthesis. Examples of suchmolecules include PNA in which the backbone is formed by peptide bonds.There is no limitation on the number of nucleotides in thepolynucleotide of the present invention. The term polynucleotide isequivalent to the term nucleic acid. On the other hand, the termoligonucleotide as used herein especially refers to polynucleotides witha smaller number of nucleotides among polynucleotides. Typically, theterm oligonucleotide refers to polynucleotides containing 2 to 100nucleotide residues, more typically about 2 to 50 nucleotide residues,but is not limited thereto.

The term “target nucleotide sequence” of the present invention refers tothe nucleotide sequence of the polynucleotide to be synthesized. Ingeneral, the nucleotide sequence of a polynucleotide is described fromthe 5′-side to the 3′-side of the sense strand. Further, the targetnucleotide sequence of the present invention includes not only the sensestrand, but also the nucleotide sequence of the complementary strandthereof, i.e. the antisense strand. More specifically, the term “targetnucleotide sequence” refers to at least either the nucleotide sequenceto be synthesized or the complementary strand thereof. Furthermore, thepresent invention provides a method that enables not only polynucleotidesynthesis, but amplification as well. In the case of carrying outpolynucleotide amplification based on the present invention, thenucleotide sequence to be amplified is referred to as the targetnucleotide sequence. The target nucleotide sequence may be the arbitraryconsecutive nucleotide sequence selected from within a longpolynucleotide, or the full-length of a single-stranded or cyclicpolynucleotide.

Furthermore, synthesis refers to the act of increasing a single targetnucleotide sequence by at least two folds or more. On the other hand,when a continuously new target nucleotide sequence is synthesized basedon the synthesized target nucleotide sequence, this is specificallyreferred to as amplification. Amplification can also be referred to ascontinuous synthesis.

Moreover, in the present invention, the provision of a starting pointfor complementary strand synthesis refers to the hybridization of the3′-end of a polynucleotide that functions as a primer required forcomplementary strand synthesis to a polynucleotide that serves as atemplate. When a starting point for complementary strand synthesis isprovided at a specific region, this means that the polynucleotide ishybridized so that the 3′-end that is to serve as the starting point forcomplementary strand synthesis is located at an arbitrary locationwithin the region. At this time, the portion of the nucleotide sequencerequired for hybridization may also be arranged outside that regionprovided the 3′-end is located in the target region.

On the other hand, the above-mentioned DNA polymerase catalyzes acomplementary strand synthesis reaction that uses as a starting pointeach of the primers annealed to the above template polynucleotide aswell as its own 3′-end under conditions that enable template-dependentcomplementary strand synthesis. In the present invention, “conditionsthat enable template-dependent complementary strand synthesis” refer toa reaction in which a new polynucleotide chain comprising a nucleotidesequence complementary to the nucleotide sequence of the templatepolynucleotide is synthesized by using as a starting point the 3′-end ofthe polynucleotide annealed to the template. In the present invention, anew polynucleotide chain may also be a molecule that differs from thetemplate or a molecule that is the same as the template. For example, anew polynucleotide chain produced by a complementary strand synthesisreaction that proceeds by annealing the 3′-end of a polynucleotide toitself, is the same molecule as the template polynucleotide. In thepresent invention, conditions that enable template-dependentcomplementary strand synthesis may also simply be referred to asconditions that enable complementary strand synthesis.

Such reactions can normally be conducted in a buffer that provides theoptimum reaction conditions for the DNA polymerase. A protective agentthat protects the activity of the DNA polymerase, inorganic saltsrequired to maintain the activity and so forth may also be presenttogether in the buffer. Such reaction conditions can be suitablyselected by a person with ordinary skill in the art according to the DNApolymerase.

In the present invention, the terms “3′-end” and “5′-end” do not referto the nucleotide of either terminus, but rather, refer to a regionlocated at the terminus that includes the single end nucleotide. Morespecifically, 500 nucleotides, preferably 100 nucleotides, or at least20 nucleotides from either terminus are included in the terms “3′-end”and “5′-end”. In contrast, in order to indicate a single end nucleotideor a nucleotide at a specific location present in the vicinity of aterminus, the numerical value of the location thereof is specified.

The target nucleotide sequence of the present invention comprises atleast a pair of complementary nucleotide sequences. The terms“identical” and “complementary” as used herein encompass cases that arenot completely identical and not completely complementary. Morespecifically, a sequence identical to a certain sequence also includes asequence complementary to a nucleotide sequence that can anneal to thecertain sequence. On the ocher hand, a complimentary sequence refers toa sequence that anneals under stringent conditions, and provides a3′-end as the starting point for complementary strand synthesis. Morespecifically, a nucleotide sequence that has an identity of typically50% to 100%, normally 70% to 100%, and preferably 80% to 100% to acertain nucleotide sequence can be mentioned as being a sequence that issubstantially identical. Identity can be determined based on knownalgorithms such as BLAST.

A template polynucleotide of the present invention is capable of forminga loop when the above complementary nucleotide sequence hybridizes.After the complementary nucleotide sequence is hybridized, it isdifficult for new base pair bonds to occur under conditions under whichbase pairing is kept stable. On the other hand, a loop is able to formnew base pair bonds with a different polynucleotide composed of anucleotide sequence complementary to the nucleotide sequence thatcomposes this loop. The nucleotide sequence that composes the loop isarbitrary.

The term “hybridize” used in the present invention refers to the bondingof a polynucleotide comprising a complementary nucleotide sequence bybase pairing. The polynucleotide that undergoes base pairing may be adifferent molecule or the same molecule. If hybridization that hasoccurred between different molecules is terminated, dissociation into aplurality of polynucleotide molecules will occur. On the other hand, apolynucleotide that has hybridized on the same molecule remains as onepolynucleotide even if the base pair bonds are dissolved. In the presentinvention, the term “anneal” is also used. There are cases in particularin which the term anneal is used when a polynucleotide hybridizes to apolynucleotide comprising a complementary nucleotide sequence, andprovides a 3′-end that serves as the starting point for complementarystrand synthesis.

The template polynucleotide of the present invention is capable offorming a loop by the annealing of its 3′-end to itself. This 3′-endannealed to itself is able to serve as a starting point forcomplementary strand synthesis using itself as template. There are norestrictions on the nucleotide sequence for annealing, provided itallows complementary strand synthesis from its 3′-end. Morespecifically, for example, 100-200 nucleotides, normally 50-80nucleotides, and preferably 20-30 nucleotides, from the 3′-end of apolynucleotide, comprise a nucleotide sequence that is complementary toan arbitrary region within the above target nucleotide sequence. At thistime, the nucleotides of the annealed 3′-end are preferably completelycomplementary to the target nucleotide sequence. Although it is notessential for the nucleotides of the 3′-end to be completelycomplementary, this is an important condition for efficientcomplementary strand synthesis.

The template polynucleotide of the present invention is capable offorming a loop by the annealing of its 3′-end to itself. Similar to theabove loop, this loop is composed of an arbitrary nucleotide sequence,and is present in a state that enables base pairing with anotherpolynucleotide.

In addition, the template polynucleotide of the present invention canhave on its 5′-end a nucleotide sequence that is complementary to anarbitrary region in the template itself. When a complementary strand ofsuch a template polynucleotide is synthesized, the 3-end of thesynthesized new polynucleotide is able to serve as a starting point of acomplementary strand synthesis reaction that uses itself as a templateby annealing to its own arbitrary region. The annealing of the 3′-end toitself forms a loop. A primer in the present invention can anneal to aloop formed in this manner.

In this manner, a product of complementary strand synthesis can bereused as a template polynucleotide by making the 5′-end of a templatepolynucleotide, a nucleotide sequence that is complementary to anarbitrary region in the template. Thus, such a template polynucleotidehas a preferable structure for achieving a highly efficientcomplementary strand synthesis reaction in the present invention.

The template polynucleotide in the present invention can be synthesizedenzymatically or chemically. For example, a template polynucleotide canbe synthesized by conducting the following steps a) through d). Thefollowing steps a) through d) can be said to be a polynucleotidesynthesis method for the LAMP method:

a) annealing a first primer to a target nucleotide sequence, andconducting a complementary strand synthesis reaction using this as astarting point, wherein said first primer (i) can provide at the 3′-enda starting point for complementary strand synthesis to a region thatdefines the 3′-side of one of the strands that compose the targetnucleotide sequence, and (ii) has on its 5′-side a nucleotide sequencecomplementary to an arbitrary region of a complementary strand synthesisreaction product that uses this primer as a starting point;

b) placing, in a condition that allows base pairing, the region to wherea second primer is to anneal in the elongation product of the firstprimer synthesized in step a), wherein said second primer (i) has on its3′-end a nucleotide sequence providing a starting point forcomplementary strand synthesis to a region that defines the 3′-side of atarget nucleotide sequence in the elongation product that uses the firstprimer as a starting point, and (ii) has on its 5′-side a nucleotidesequence that is complementary to an arbitrary region of a complementarystrand synthesis reaction product that uses this primer as an startingpoint;

c) annealing said second primer to the region that can form base pairingin step b); and carrying out complementary strand synthesis using thisas a starting point; and,

d) annealing the 3′-end of the elongation product of the second primersynthesized in step c) to itself, and carrying out complementary strandsynthesis using itself as template.

The following provides more specific explanation of the above stepsbased on FIG. 1. In the following explanation, an example is shown of aprocess in which a template polynucleotide is produced in the presentinvention using a first primer (RA) comprising R2 and R1c and a secondprimer (FA) comprising F2 and F1c. In the following explanation, thefirst primer and the second primer are tentatively named RA and FA,respectively.

The first primer RA (i) can provide at the 3′-end a starting point forcomplementary strand synthesis to a region that defines the 3′-side ofone of the strands that compose a target nucleotide sequence, and (ii)has on the 5′-side a nucleotide sequence that is complementary to anarbitrary region of a complementary strand synthesis reaction productthat uses this primer for an starting point. The region on the 3′-sideof RA is referred to as R2, while the region on the 5′-side is referredto as R1c. On the other hand, the second primer FA (i) has in its 3′-enda nucleotide sequence that is capable of providing a starting point forcomplementary strand synthesis to a region that defines the 3′-side of atarget nucleotide sequence in an elongation production that uses theabove first primer RA as a starting point, and (ii) on the 5′-side anucleotide sequence that is complementary to an arbitrary region of acomplementary strand synthesis product that uses this primer as anstarting point. The 3′-side of FA is referred to as F2, while the5′-side is referred to as F1c. Moreover, each region that composes the3′-side and 5′-side of RA and FA respectively comprises a nucleotidesequence complementary to the following regions.

3′-side of RA (R2): R2c

5′-side of RA (R1c): R1

3′-side of FA (F2): F2c

5′-side of FA (F1c): F1

Ultimately, the structure of RA is determined by R2c and R1 of thetarget nucleotide sequence, while the structure of FA is determined byF2c and F1 in the target nucleotide sequence. Thus, in the presentinvention, the target nucleotide sequence is required to be a nucleotidesequence in which at least a portion of that nucleotide sequence iseither known or is predictable. The portions for which the nucleotidesequence is to be clarified, comprise each of the regions that determinethe structures of RA and FA, or regions comprising their complementarynucleotide sequences. R2c and R1c (or F2c and F1c) may be linked to eachother or may exist separately. The state of the loop portion formed byself-annealing of the product polynucleotide depends on the relativeposition of the two regions. The term self-annealing means that a regioncomprising the 3′-end of a single-stranded polynucleotide hybridizes tothe complementary nucleotide sequence of the polynucleotide itself tostart complementary strand synthesis using the polynucleotide itself astemplate. The two regions are preferably not unnecessarily apart toachieve self-annealing of the product polynucleotide more preferentiallythan annealing of two molecules. Thus, a preferred length of the spacernucleotide sequence between the two regions is typically 0 to 500nucleotides. However, in some cases, regions existing too close to eachother may be disadvantageous for forming a desirable loop byself-annealing.

More specifically, the loop preferably has a structure that enables theannealing of a new oligonucleotide and a smooth start for thecomplementary strand synthesis reaction accompanying stranddisplacement. Thus, more preferably, the primers are designed so thatthe distance between region R2c and region R1c located on the 5′-side ofX2c is 0 to 100 nucleotides, more preferably it is 10 to 70 nucleotides.This value does not include the length of R1c and R2c. The nucleotidelength of the loop portion further includes the length corresponding toR2. Also, similar conditions are applied to the distance between regionF2c and region F1c in FA.

Regions R2 and R1c (or F2 and F1c) constituting RA and FA are typicallyarranged continuously without overlapping with respect to the abovenucleotide sequence. Alternatively, if R2 (or F2) and R1c (or F1c) sharea common nucleotide sequence, they may be placed so that they partlyoverlap. R2 (or F2) should be placed at the 3′-end at all times since ithas to function as a primer. On the other hand, R1c (or F1c) is placedat the 5′-end as described below, to provide a function as a primer tothe 3′-end of the complementary strand synthesized using R1c (or F1c) astemplate. The complementary strand obtained using the oligonucleotide asthe synthesis origin serves as the template of the reverse complementarystrand synthesis in the next step, and finally, RA and FA are alsocopied as the template to the complementary strand. The copied 3′-endcontains the nucleotide sequence R1 (or F1), and anneals to R1c (or F1c)located within the same strand to form a loop.

To begin with, complementary strand synthesis is carried out byannealing R2 of the first primer to R2c in the target nucleotidesequence (FIG. 1-(1)). When the elongation product of the first primeris converted to a single strand, and complementary strand synthesis iscarried out by annealing F2 of the second primer to its F2c,complementary strand synthesis ends when it has reached the 5′-end ofthe first primer. The elongation product of the second primersynthesized at this time has R1 in its 3′-end. R1 of the 3′-end is aregion synthesized using R1c of the 5′-side of the first primer astemplate. R1 serves as the starting point for complementary strandsynthesis by annealing to its own R1c, and complementary strandsynthesis is conducted using itself as template (see “recyclingproducts” of FIG. 1-(2)).

The polynucleotide that is produced as a result of the above reactionhas a set of complementary nucleotide sequences composed of the targetnucleotide sequence and its complementary strand, and a loop thatenables base pairing is formed when they hybridize. In addition, F1comprises in its 3′-end a nucleotide sequence complementary to its ownF1c. F1 is a region that is synthesized by using F1c of the secondprimer as template. Namely, this product is none other than a templatepolynucleotide in the present invention.

Furthermore, the structures for providing a polynucleotide that canserve as a new starting material for a polynucleotide synthesis reactionis shown as “recycling products” in FIG. 1. The products that areproduced by using these structures as templates are all capable ofproducing new template polynucleotides.

Furthermore, in FIG. 1, a step is shown in which complementary strandsynthesis is carried out by a template polynucleotide that has beenproduced by the further annealing of its 3′-end to itself. As a result,a template polynucleotide having two sets of complementary targetnucleotide sequences is produced (FIG. 1-(3)). Furthermore, the sequencewhich composes a target nucleotide sequence in FIG. 1-(3) is anucleotide sequence that composes between F2 and R2c and between itscomplementary nucleotide strands R2 and F2c.

The above reaction actually proceeds in parallel even in thecomplementary strand of the target nucleotide sequence. Namely, thereaction starts from complementary strand synthesis of the secondprimer, proceeds through the elongation product of the first primer(FIG. 1-(2′)), and produces a template polynucleotide having two sets ofcomplementary target nucleotide sequences (FIG. 1-(3′)). The templatepolynucleotide shown in FIG. 1-(3) and the template polynucleotide shownin FIG. 1-(3′) have mutually complementary nucleotide sequences.

Next, in carrying out step b) in a reaction for synthesizing a templatepolynucleotide based on the LAMP method, namely the step of placing, ina condition that allows base pairing, the region to which the secondprimer in an elongation product of the first primer synthesized in stepa) is to anneal, it is advantageous to use an outer primer. In thepresent invention, outer primers refer to a primer comprising anucleotide sequence that is complementary to the upstream from the firstprimer and second primer that are annealed to the target nucleotidesequence. In the present invention, upstream refers to the 3′-side inthe template. Thus, the regions that are annealed by an outer primer areregions on the 5′-sides as viewed from the first primer and secondprimer.

For example, FIG. 1-(1) describes an outer primer R3 that anneals to R3clocated on the 3′-side of region R2c to which RA anneals. Similarly, inits complementary strand, outer primer F3 can anneal to F3c located onthe 3′-side of the region to which FA anneals. An oligonucleotide havinga nucleotide sequence that functions as a primer at least on its 3′-sidecan be used for the outer primer. Both the first primer and secondprimer can be used as two of the three types of primers in the presentinvention. On the other hand, the outer primer described here does notnecessarily compose the three types of primers of the present invention.The outer primer is used to synthesize a template polynucleotide.

In contrast to the first primer and second primer that are normallycomposed of a combination of two primers, the outer primer may becomposed of an arbitrary number of primers. In the present invention, atypical outer primer consists of two outer primers capable of providinga starting point for complementary strand synthesis to the upstream ofeach of the first primer and second primer. However, the method of thepresent invention can be practiced even in the case where an outerprimer is arranged only to either the first primer or the second primer.Alternatively, a plurality of outer primers may be combined with each orone of the first primer and second primer. In any case, whenaccompanying complementary strand synthesis from farther upstream, aproduct of a complementary strand synthesis reaction that uses the firstprimer and second primer as replication origin can be producedefficiently.

Complementary strand synthesis from an outer primer in the presentinvention is designed so that it begins later than the complementarystrand synthesis using the first and second primers as replicationorigin. The simplest method for accomplishing this is to make theconcentrations of the first and second primers higher than that of theouter primer. More specifically, normally by using primers having adifference in concentrations of 2-50 times, and preferably 4-10 times,complementary strand synthesis from the first and second primers can bepreferentially conducted. In addition, the timing of synthesis can alsobe controlled by setting the melting temperature (Tm) of the outerprimer to be lower than Tm of the first and second primers.

Namely, by designing the Tm of the outer primer to (outer primerF3:F3c)≦(F2c/F2) with respect to inner primer FA, nucleic acidamplification can be carried out efficiently. It is preferable to designeach region that composes FA so that annealing between (F1c/F1) takesplace preferentially to (F2c/F2). In the design, Tm, the constituentnucleotides, and so forth are taken into consideration. Moreover, thehigh possibility that the annealing between F1c/F1 will proceedpreferentially since it is an intramolecular reaction, is also takeninto consideration. Similar conditions are also considered in the designof RA that anneals to the FA elongation product.

As a result of creating such a relationship, stochastically idealreaction conditions can be achieved. Melting temperature (Tm) cantheoretically be calculated if other conditions are constant by thelength of the complementary strand that is annealed and the combinationof nucleotides that compose base pairs. Thus, a person with ordinaryskill in the art is able to easily arrive at the preferable conditionsbased on the teachings of the present description.

Moreover, in order to adjust the timing of annealing of outer primer, aphenomenon referred to as “contiguous stacking” can be applied.Contiguous stacking is a phenomenon in which an oligonucleotide that isunable to anneal alone can be made to anneal by placing it adjacent to adouble-stranded portion (Chiara Borghesi-Nicoletti et al., BioTechniques 12, 474-477, 1992). In other words, the outer primer is madeplaced adjacent to a first primer and second primer, and designed sothat the outer primer is unable to anneal alone under the incubationconditions. When this is done, since it is possible for the outer primerto anneal only after the first and second primers have annealed,annealing of the first and second primers inevitably precedes. Based onthis principle, an example of setting the nucleotide sequence of anoligonucleotide required as a primer for the series of reactions isdescribed in the Examples.

In the above reaction, a polynucleotide sample that contains a targetnucleotide sequence can use an arbitrary polynucleotide that is capableof serving as a template of complementary strand synthesis. Specificexamples can include DNA, RNA, as well as their derivatives and chimericmolecules. Moreover, in addition to natural polynucleotides such asgenome DNA and mRNA, polynucleotides artificially integrated intoplasmids, phages, or such may also be used for the polynucleotidesample. The polynucleotide sample may be used in not only the purifiedstate, but also the crude state. Polynucleotides present within cellsare also applicable to in situ synthesis.

Next, in the present invention, at least three types of primers are usedthat are capable of providing a starting point for complementary strandsynthesis to different regions of a loop. The loops to which the primersimpart the starting points of complementary strand synthesis includesnot only the loops present in the above template polynucleotides, butalso loops formed by new polynucleotides produced as a result ofcomplementary strand synthesis using their 3′-ends or any of the primersas a starting point.

A template polynucleotide has at least the following two loops:

-   1. a loop formed by the hybridization of a target nucleotide    sequence; and,-   2. a loop formed by the annealing of the 3′-end of a template    polynucleotide to itself.

In the case where the template polynucleotide has two or more sets ofcomplementary nucleotide sequences, and the complementary nucleotidesequences are alternately arranged, loops are formed on the templatepolynucleotide corresponding to the number of complementary nucleotidesequences. For example, a polynucleotide is assumed that is composed ofa certain nucleotide sequence A and its complementary nucleotidesequence a. An example of the state in which a plurality of sets ofcomplementary nucleotide sequences is alternately arranged, can berepresented as shown below.5′-[A]-(L)-[a]-[A]-(L)-[a]-[A]-(L)-[a]-3′

In this example, three loops can be formed when hybridization occursbetween adjacent [A] and [a]. The portions where loops are formed due tohybridization of complementary nucleotide sequences are indicated with-(L)-. Moreover, there is an additional loop formed by annealing of the3′-end to itself, resulting in a total of four loops being formed bythis template polynucleotide. When the 3′-end forms a loop by annealingto itself, a portion of the nucleotide sequence of [a] is used forannealing. Thus, [a] of the 3′-end has a region that is a nucleotidesequence complementary to [A] while being used for annealing to itself.

Furthermore, there are no restrictions on the number of complementarynucleotide sequences that can be present in a template polynucleotide ofthe present invention. Thus, a template polynucleotide composed of [A]and [a] can be represented by a formula like that shown below. In thisformula, n refers to any natural number.5′-{[A]-(L)-[a]}n-3′

On the other hand, a loop formed by a new polynucleotide produced as aresult of complementary strand synthesis that uses the 3′-end of atemplate polynucleotide or any of the primers as a starting point isspecifically formed in the following manner. To begin with, acomplementary strand synthesis reaction can be carried out for thetemplate polynucleotide by annealing its 3′-end to itself and usingitself as template. This reaction proceeds continuously if allowed to bein a state in which base pairing can be repeated between the 3′-end andthe region to which it anneals. As a result, the nucleotide sequencecomplementary to the template polynucleotide is repeatedly elongated.Thus, in the case where the template polynucleotide is composed of oneset of complementary nucleotide sequences, the number of complementarynucleotide sequences in the new polynucleotide obtained by complementarystrand synthesis doubles with each repetition of the complementarystrand synthesis reaction. Here, when two sets of complementarynucleotide sequences are respectively hybridized in the newly producedpolynucleotide, a number of loops occur that corresponds to the numberof sets of complementary nucleotide sequences (refer to, for example,FIG. 1-(4) or FIG. 1-(5)). Among these loops, some of the loops arethose originally present in the template polynucleotide. Other loops arecomposed of loop complementary nucleotide sequences of the templatepolynucleotide. New loops produced in this manner are also included inthe loops to which a primer anneals in the present invention.

Moreover, a loop formed by a new polynucleotide produced by theannealing of a primer to a loop present in a template polynucleotide andby using this as a starting point for complementary strand synthesis, isalso included in the loops of the present invention. For example, acomplementary strand can be synthesized by using as a starting point aprimer capable of providing a starting point for complementary strandsynthesis to a loop formed by annealing of the 3′-end of a templatepolynucleotide to itself. This product becomes a polynucleotidecomprising a nucleotide sequence complementary to the template. Similarto template polynucleotide, this polynucleotide forms a loop byhybridization with a complementary nucleotide sequence. Namely, apolynucleotide is produced that has a loop comprising a nucleotidesequence that is complementary to a loop present in the templatepolynucleotide.

A primer capable of providing a starting point for complementary strandsynthesis to a new loop formed in this manner is included in the primersof the present invention. Alternatively, as was previously described, inthe case where a template polynucleotide contains a nucleotide sequencecomplementary to an arbitrary region on its own 5′-end, the 3′-end ofpolynucleotide synthesized by using this as a template is capable offorming a loop by annealing to itself. In the present invention, aprimer can also be used that is capable of providing a starting pointfor complementary strand synthesis to a loop formed in this manner.

Furthermore, when the 5′-end of a template polynucleotide contains anucleotide sequence complementary to an arbitrary region of itself, the5′-end is able to form a loop by hybridizing to itself. A loop of the5′-end is also in a state that allows base pairing. However, normally,only a small region extending from the loop to the 5′-end is eligiblefor complementary strand synthesis from a primer that provides astarting point for complementary strand synthesis to this loop.

In the present invention, three or more types of primers of the presentinvention are used that are capable of serving as a starting point forcomplementary strand synthesis in at least one type, preferably twotypes, or three or more types of loops selected from several types ofloops as previously mentioned. “Three or more types” refers to threeprimers or more providing a starting point for complementary strandsynthesis at mutually different locations.

At least three types, preferably four types, or five or more types ofprimers can be combined in the present invention. It should be notedthat “mutually different locations” refer to the location of the 3′-endof each primer being mutually different when the primer has beenannealed. Thus, those nucleotide sequences required for annealing may bepartially overlapping. However, in the case where the regions requiredfor annealing overlap, since competition occurs between primers, it ispreferable to design the nucleotide sequences of the primers so thatthey can be annealed to mutually independent regions as much aspossible. Moreover, complementary strand synthesis can be expected toproceed more rapidly by designing the nucleotide sequences of theprimers so that there is minimal overlapping even with respect to thoseregions subject to hybridization and annealing in the templatepolynucleotide. Thus, for example, the combination of a templatepolynucleotide and three or more types of primers that anneal tomutually different loops formed in a complementary strand synthesisproduct that is produced by using the template polynucleotide astemplate can be said to be a preferable primer combination for thepresent invention.

A nucleotide sequence that composes a primer of the present inventioncontains a nucleotide sequence that is complementary to the nucleotidesequences of the above-mentioned plurality of loops. Its 3′-side isrequired to impart a starting point for complementary strand synthesisto those loops. Thus, at least its 3′-end should be located within theloops. However, all of the nucleotide sequences required for annealingneed not be arranged within the loops. Thus, as long as the primer isable to impart a starting point for complementary strand synthesis underthe required reaction conditions, a portion of the nucleotide sequencesrequired for annealing may overlap with the nucleotide sequence thatcomposes the double-strand polynucleotide adjacent to the loop. Inaddition, an arbitrary nucleotide sequence may be also added to the5′-side of the primer. In the present invention, the previouslymentioned LAMP primer can be used as a primer that provides a startingpoint for complementary strand synthesis to a loop present in a templatepolynucleotide.

The LAMP method (Nucleic Acid Res. 2000, Vol. 28, No. 12 e63, WO00/28082) is a method that allows highly isothermal complementary strandsynthesis reactions by combining a primer that serves as a startingpoint for complementary strand synthesis by annealing a templatepolynucleotide to its own 3′-end, while also being able to impart astarting point for complementary strand synthesis to a loop formed atthis time. At least two primers are used in the LAMP method.

-   [1] First primer: The first primer can provide (i) at its 3′-end a    starting point for complementary strand synthesis to a region that    defines the 3′-side of one of the strands that compose a target    nucleotide sequence, and (ii) on the 5′-side a nucleotide sequence    that is complementary to an arbitrary region of a complementary    strand synthesis reaction product that uses this primer as an    starting point,-   [2] Second primer: The second primer has on its 3′-end a nucleotide    sequence capable of providing a starting point for complementary    strand synthesis to a region that defines the 3′-side of a target    nucleotide sequence in an elongation product that uses the above    first primer as a starting point, and (ii) on the 5′-side a    nucleotide sequence that is complementary to an arbitrary region of    a complementary strand synthesis reaction product that uses this    primer as an starting point,

that is, the first primer and second primer used for synthesis of theabove template polynucleotide are able to compose a portion of at leastthe three types of primers in the polynucleotide synthesis method basedon the present invention. In other words, the present invention can becarried out by combining a third primer that is capable of providing astarting point for complementary strand synthesis at a location thatdiffers from the above first and second primers within a templatepolynucleotide or loop formed by a reaction product. In the presentinvention, in addition to the primers required for synthesis of a targetnucleotide sequence, the primer that is additionally combined isreferred to, sometimes, as a loop primer.

It is not essential that the primer used for template polynucleotidesynthesis is also used as a primer for synthesis of a target nucleotidesequence. A primer for synthesizing a target nucleotide sequence may beadded separate to the primer for synthesizing a template polynucleotide.However, it is usually more practical to compose a reaction with as fewelements as possible.

A particularly preferable loop primer in the present invention is thatwhich is capable of providing an starting point for complementary strandsynthesis to a region between R1 and R2 (or between F1 and F2). Wheneither a loop primer capable of annealing between R1 and R2 or betweenF1 and F2 is combined with a LAMP primer, three types of primers areused. Moreover; if primers capable of annealing between both R1 and R2and between F1 and F2 are combined together, in addition to the twotypes of loop primers for the LAMP primers, a total of four types ofprimers are used. In the present invention, a loop primer can beexpected to have the action of accelerating the polynucleotide synthesisreaction by combining at least one type, and preferably two or moretypes.

This loop primer anneals to a loop containing R2 (refer to, for example,FIG. 1-(4)). This loop is composed of a region extending from R1 to R2in the target nucleotide sequence (however, in FIG. 1-(4), R2 iscontained, but not R1). For such a loop, the loop primer of the presentinvention can be a nucleotide having an arbitrary nucleotide sequencecomprising a nucleotide sequence complementary to the nucleotidesequence that composes the loop.

However, as is clearly shown in the examples, a polynucleotide can besynthesized more efficiently by selecting a region within a loop. Forexample, a loop primer capable of providing a replication origin to aregion that does not overlap with R1c, namely one closer to the 3′-sidethan R1c, enhances the reaction efficiency of the LAMP methodconsiderably.

Ultimately, examples of preferable loop primers that are combined whenapplying the present invention to the LAMP method, include those primersthat satisfy the following condition: namely, those that provide astarting point for complementary strand synthesis to a loop (i) formedby a template polynucleotide and/or polynucleotide produced by areaction of template polynucleotide with a first primer and a secondprimer, and (ii) to which the first primer and the second primer cannotanneal. The loop primer should be able to anneal to a region in which a3′-end is arranged, which serves as the starting point for complementarystrand synthesis within the loop. Thus, not only in the case in whichthe nucleotide sequence required for annealing is contained completelywithin the loop, but also in the case in which a portion of thatsequence overlaps with a region other than the loop, the preferablecondition can be said to be satisfied if the 3′-end is located withinthe loop. For example, the Examples show that even when a portion of thenucleotide sequence to which the loop primer is to anneal extends to adouble-stranded structure adjacent to the loop, reaction acceleratingeffects that are similar to those seen when the region to be annealed isarranged completely within the loop can be obtained.

The preferable positional relationship between loop primer andnucleotide sequence that composes a template polynucleotide is shown inFIG. 3. FIG. 3 depicts complementary strand synthesis using the 3′-endof F1 that anneals to itself as a starting point. FIG. 3 indicates thesame structure as FIG. 1-(2′). Namely, once the complementary strandsynthesis shown in this figure is over, a template polynucleotide of thepresent invention is completed. As shown in FIG. 3, loop primer R is aprimer that anneals to a loop containing R2. It should be noted thatFIG. 3 is provided to indicate a region to which loop primer R anneals.The complementary strand synthesis from loop primer R annealed to thestructure of FIG. 3 actually ends as soon as the 5′-end is reached. Whencomplementary strand synthesis is carried out by annealing to the loopof a similar nucleotide sequence formed in the reaction product producedfrom template polynucleotide, the complementary strand synthesis thatuses loop primer R as a starting point, contributes to the improvementof reaction efficiency.

When the present invention is applied to the LAMP method, it ispreferable that the loop primers are able to anneal under the sameconditions as the conditions of the LAMP method. The fact that allreactions can be carried out without changing the temperature is a majoradvantage of the LAMP method. When combining loop primers for thepresent invention as well, it is ideal to use loop primers under thesame temperature conditions as the LAMP method so as not to lose thisadvantage. In order to accomplish this, the Tm of the loop primers isdesigned to be at the same level as the primers for the LAMP method. Tmis determined by the types and number of nucleotides that compose theprimers as well as the salts and various types of components that havean effect on Tm contained in the reaction solution. Thus, in the presentinvention, Tm of the loop primers is adjusted by the nucleotidesequences of the primers to match the conditions of the reactionsolution for the LAMP method. One skilled in the art is able to impart asuitable Tm by adjusting the nucleotide sequences of the primers tomatch the various conditions. These conditions relating to the loopprimers are also applied to the loop composed by F1c and F2c (or by R1cand R2c).

The structure shown in FIG. 1-(4) is the product produced as a result ofrepeating a complementary strand synthesis reaction in which thetemplate polynucleotide uses itself as a template. That loop is composedof a nucleotide sequence that is complementary to the loop of thetemplate polynucleotide. Primers that anneal to the templatepolynucleotide are unable to anneal as primers to a nucleotide sequencecomplementary to the template polynucleotide. In other words, apreferable loop primer in the present invention is able to impart astarting point for complementary strand synthesis to a loop formed on anew polynucleotide that was produced by a synthesis using a templatepolynucleotide as template. As a result of employing this relationship,complementary strand synthesis that uses a template polynucleotide astemplate and complementary strand synthesis that uses a newly producedpolynucleotide as template proceed. As a result, more polynucleotidesare rapidly produced in a short period of time.

As already described above, the 3′-side of a loop primer of the presentinvention serves as a starting point for complementary strand synthesisby annealing to the loop. In contrast, it is possible to arrange on the5′-side of a loop primer, a nucleotide sequence that is complementary toa nucleotide sequence that composes an arbitrary region of acomplementary strand synthesized by using the 3′-end of the loop primeras a starting point. In the present invention, a preferable example of anucleotide sequence for the nucleotide sequence arranged on the 5′-sideof a loop primer, is a nucleotide sequence substantially identical tothe nucleotide sequence arranged on the 5′-side of the previouslymentioned inner primer (F1c or R1c). Herein, substantially identicalsequences include cases in which the 3′-end of a complementary strandproduced by using a nucleotide sequence arranged on the 5′-side of aloop primer as template is able to provide a starting point forcomplementary strand synthesis to a region to which is annealed the3′-end of a complementary strand produced by using the nucleotidesequence arranged on the 5′-side of the above inner primer RA or FA astemplate.

For example, a nucleotide sequence substantially identical to thenucleotide sequence R1c arranged on the 5′-side of RA can be arranged inloop primer R that anneals to a loop containing the nucleotide sequenceof primer RA. Similarly, a nucleotide sequence substantially identicalto the nucleotide sequence that composes F1c can be arranged on the5′-side in loop primer F that anneals to a loop that contains thenucleotide sequence of inner primer FA.

An elongation product produced by a loop primer having a nucleotidesequence complementary to itself on the 5′-side is able to impart anucleotide sequence complementary to itself on the 3′-end of acomplementary strand produced by using this as template. As a result,products of loop primers also have structures that function as LAMPrecycling products. Thus, the LAMP nucleic acid amplification reactionis also triggered by a loop primer, making it possible to anticipate ahigh amplification rate.

As is clear from the above explanation, when applying the LAMP method tothe polynucleotide synthesis method of the present invention, if anabove-mentioned first primer, second primer, and loop primer are used, atemplate polynucleotide can be obtained based on a target nucleotidesequence, and moreover, the polynucleotide synthesis method according tothe present invention can be carried out. In other words, if thefollowing components are incubated together with a target nucleotidesequence, the polynucleotide synthesis method of the present inventioncan be carried out. These reaction components may be mixed in any orderor mixed simultaneously. By further adding an outer primer (to bedescribed later) to this reaction system, all steps can be carried outat the same temperature, targeting a double-stranded polynucleotidesample.

-   -   a polynucleotide sample containing a target nucleotide sequence    -   a first primer    -   a second primer    -   a loop primer (one type, or two or more types)    -   a DNA polymerase capable of catalyzing complementary strand        synthesis accompanying strand displacement    -   a substrate for complementary strand synthesis

Moreover, if these components are incubated under conditions that allowcomplementary strand synthesis, the polynucleotide ultimatelysynthesized based on the target nucleotide sequence will continue toproduce a new template polynucleotide. All the newly producedpolynucleotides are used as materials for synthesizing newpolynucleotides. Namely, polynucleotide amplification occurs. Thepresent invention includes polynucleotide amplification methods achievedin this manner. Amplification efficiency can be improved considerably byamplifying polynucleotides based on the polynucleotide synthesis methodof the present invention. For example, by carrying out an amplificationmethod based on the present invention combining a loop primer with aLAMP primer under preferable conditions, the reaction time can beshortened to half or less compared to when a loop primer is not present.

The following provides an explanation of a reaction in which adouble-stranded polynucleotide is amplified by using RA (first primer),FA (second primer), loop primer F, and loop primer R and combining witha DNA polymerase that catalyzes a strand displacement type ofcomplementary strand synthesis reaction, also referring to the basicprinciple illustrated in FIGS. 1 and 2. In this example, RA and FAcompose a set consisting of a first primer and second primer forsynthesizing a target nucleotide sequence. Loop primer F is capable ofproviding a starting point for complementary strand synthesis to a loopcontaining F2, while loop primer R is capable of providing a startingpoint for complementary strand synthesis to a loop containing R2.

As already described, a template polynucleotide of the present inventionis obtained when the step of conducting complementary strand synthesisby annealing the 3′-end shown in FIG. 1-(2) or FIG. 1-(2′) to itself iscompleted. Moreover, in the loop of this template polynucleotide,complementary strand synthesis is carried out by annealing by RA (FIG.1-(2)) or FA (FIG. 1-(2′)), respectively, and the 3′-end of templatepolynucleotide once again becomes a single strand due to displacement.After becoming a single strand, the 3′-end anneals to itself andcomplementary strand synthesis proceeds. As a result, the products shownin FIG. 1-(3) and FIG. 1-(3′) are produced. In this manner, the cycle ofcomplementary strand synthesis consisting of annealing of primer RA orprimer FA to a loop, complementary strand synthesis, displacement of the3′-end of the template polynucleotide, and finally, annealing of the3′-end to itself, is repeated.

At this time, as a result of the elongation of the 3′-end of thetemplate polynucleotide, which uses itself as template, the product ofcomplementary strand synthesis that uses RA or FA annealed to the loopas a replication origin is displaced, resulting in the production of anew single-stranded polynucleotide. This single-stranded polynucleotidehas nucleotide sequences complementary to itself on its 3′-end and5′-end. In other words, products that have a structure identical to FIG.1-(2) and FIG. 1-(2′) are made. These become new templatepolynucleotides and a similar reaction proceeds repeatedly. The entirereaction mechanism up to this point has been clarified as the LAMPmethod.

In FIG. 1-(3), primer FA annealed to the loop elongates while displacingthe double-stranded structure of the template polynucleotide until itfinally reaches the 5′-end. At this time, the 3′-end side of thedisplaced template polynucleotide has a single-stranded structure.Consequently, nucleotide sequences that are complementary to themselvesare mutually hybridized. The product formed in this manner has thestructure shown in FIG. 1-(4). Namely, a loop is formed by annealing the3′-end of the template polynucleotide to itself simultaneous tohybridization of complementary nucleotide sequences. The loop formed dueto hybridization of complementary nucleotide sequences is acomplementary strand that is a copy of the loop portion of the templatepolynucleotide used as the original. As is clear from the drawings, thisloop contains R2, and FA and RA are unable to anneal to it. However,loop primer R is able to anneal to the loop containing R2. Therefore,complementary strand synthesis begins by annealing loop primer R to theloop that contains R2.

On the other hand, a polynucleotide amplification reaction based on theknown LAMP method continues. Namely, the amplification reaction due tothe elongation reaction by the template polynucleotide that uses itselfas template and by primer FA that anneals to the loop, continues. Thisreaction itself is none other than a polynucleotide amplificationreaction in which new template polynucleotides are continuouslyproduced. It should be noted that the amplification reaction starting inFIG. 1-(4) that has previously been clarified with the LAMP method isnot shown herein.

Accompanying elongation of loop primer R, both template polynucleotideand the elongation product of primer FA are displaced to enable basepairing, again resulting in the production of a template polynucleotide.The elongation product of loop primer R in particular shown in FIG.1-(6) has a structure that allows annealing of three types of primersconsisting of loop primer F, loop primer R, and FA. This type of productcannot be produced with the known LAMP method. The method of the presentinvention produces the template polynucleotide shown in FIG. 1-(6)separate to the known product based on the LAMP method. The templatepolynucleotide shown in FIG. 1-(6) starts an additional polynucleotideamplification reaction together with a loop primer. In this manner, thereaction in which a new polynucleotide is further produced, proceedsseparately from the complementary strand synthesis known in the LAMPmethod. As a result, the synthesis efficiency of complementary strandsynthesis is dramatically improved, and the reaction time can beconsiderably shortened.

Furthermore, in the structure shown in FIG. 1-(6), primer RA is able toanneal to the region containing R2c near the 5′-end. However, since aprimer that anneals to this location is only eligible for complementarystrand synthesis with respect to the small region to the 5′-end, it isnot shown in FIG. 1-(6).

The following description continues to provide an explanation of thetype of products that are produced when the reaction of the presentinvention is allowed to continue further. The elongation productproduced from loop primer R in FIG. 1-(4) has the structure shown inFIG. 1-(6). In FIG. 1-(6), a structure is shown in which two loopprimers and one primer FA anneal in the same manner as FIG. 1-(5). Amongthese, the elongation products of the two loop primers are completelydisplaced to produce a single-stranded polynucleotide by a complementarystrand synthesis reaction from the 3′-end in which the polynucleotideshown in FIG. 1-(6) itself is used as a template.

On the other hand, complementary strand synthesis in which thepolynucleotide shown in FIG. 1-(6) uses itself as template is completed.Complementary strand synthesis starts in the loop including F2ccomprising FIG. 2-(7) by annealing primer FA to that loop. Since theloop containing F2c comprising FIG. 2-(7) is actually already formed inFIG. 1-(6), a reaction in which FA anneals to this loop proceeds inparallel with the reaction that produces FIG. 2-(7). In any case, theregion extending from the polynucleotide loop shown in FIG. 2-(7) to the3′-end is displaced resulting in a single strand due to elongation ofthe FA annealed to the loop.

The complementary nucleotide sequences contained in the region that hasbecome a single strand are respectively hybridized, resulting in thestructure shown in FIG. 2-(8). Furthermore, there is a high possibilitythat hybridization with complementary nucleotide sequences in physicallyclose regions will preferentially occur in the polynucleotide that hasbecome a single strand. Thus, hybridization with an adjacent regiontakes precedence over hybridization with other molecules such as variousprimers or complementary nucleotide sequences located farther away. Asis clear from the drawings, loop primer R anneals to the 3′-end of thepolynucleotide shown in FIG. 2(8). In addition, two loops are formed towhich primers FA and RA are able to anneal. In addition to theelongation of each primer that has annealed to the loop, they are alsodisplaced by an elongation reaction from loop primer R resulting in asingle-stranded polynucleotide, which dissociate from the polynucleotideof FIG. 2-(8).

The double-stranded polynucleotide formed as a result of complementarystrand synthesis by loop primer R does not form any new loops, andbecomes the stable double-stranded polynucleotide of FIG. 2-(12).Although this polynucleotide is stable and is a double-strandedpolynucleotide under the reaction conditions of the LAMP method, it canbe used as a new template. In other words, even though it is adouble-stranded polynucleotide unaccompanied by a loop as shown in FIG.2-(12), it is capable of starting a new reaction by serving as atemplate for FA and RA. The structure shown in FIG. 2-(12) does not haveregions R3 or F3 (FIG. 1-(1)) to which an outer primer is to anneal,which was used when the double-stranded polynucleotide was used as atemplate polynucleotide. However, in the structure shown in FIG. 2-(12),since a plurality of regions to which primers FA and RA are able toanneal are arranged consecutively, the upstream FA can be expected todemonstrate the function of an outer primer for downstream FA. Thereaction in which double-stranded polynucleotide is used as a templateis described later.

The single-stranded polynucleotide produced by using the polynucleotideof FIG. 2-(8) as template has the three types of structures shown inFIG. 2-(9), FIG. 2-(10), and FIG. 2-(11). The relationships between thetemplates and primers that yield each structure are indicated below.Each polynucleotide is indicated by the number in parenthesis ( ) inFIG. 2.

Template (7) (8) (8) Primer FA RA FA Single strand polynucleotide (9)(10) (11)

The following reaction proceeds from these single-strandedpolynucleotides as a result of the annealing of primers corresponding tothe loop formed by each polynucleotide to the polynucleotide. To beginwith, polynucleotide of FIG. 2-(9) yields the products shown in FIG.2-(14) and FIG. 2-(15) as elongation products by primer FA annealing toF2c and primer RA annealing to R2c. These products both dissociate assingle-stranded polynucleotides as a result of being displaced due tothe complementary strand synthesis reaction from loop primer R annealedto a loop containing R2 of polynucleotide FIG. 2-(9) The twopolynucleotides shown in FIG. 2-(14) and FIG. 2-(15) have a structureequivalent to the series of polynucleotides of FIG. 1-(2) and FIG. 1-(3)indicated as “recycling products” in FIG. 1. A strict comparison revealsthat the arrangement of R2 and F2 (or R2c and F2c) within the loop whenthe loop is formed, differs between the two. However, thesepolynucleotides both lead to the production of template polynucleotidesand compose a polynucleotide amplification reaction.

Next, attention is focused on polynucleotide of FIG. 2-(10). Elongationproducts of primer FA and primer RA are also produced from thesepolynucleotides as single-stranded polynucleotides of FIG. 2-(15′) andFIG. 2-(16) in nearly the same manner as FIG. 2-(9). Thesepolynucleotides are displaced accompanying complementary strandsynthesis of loop primer R, and dissociate as single-strandedpolynucleotides. The polynucleotide shown in FIG. 2-(15′) has astructure equivalent to FIG. 1-(3′). FIG. 2-(16) is also a templatepolynucleotide having a 3′-end capable of annealing to itself in thesame manner as FIG. 1-(3). Thus, both function as templatepolynucleotides or as templates for new complementary strand reactions.

Moreover, polynucleotide of FIG. 2-(11) also produces a startingmaterial for a new reaction in the same manner as the abovepolynucleotide. Namely, FIG. 2-(17) and FIG. 2-(18) are respectivelyproduced as elongation products of primer FA and primer RA that annealto the loop. These products are displaced accompanying complementarystrand synthesis of loop primer R, and dissociate as single-strandedpolynucleotides. The produced FIG. 2-(18) is none other than a templatepolynucleotide having a 3′-end capable of annealing to itself in thesame manner as FIG. 1-(3), while FIG. 2-(17) is none other than templatepolynucleotide provided with a 3′-end capable of annealing to itself inthe same manner as FIG. 1-(3′).

FIG. 2-(13) derived from loop primer F is thought to undergo reactionssuch as complementary strand synthesis followed by annealing of primerFA, complementary strand synthesis from loop primer R that anneals tothe region displaced by the above complementary strand synthesis, andthe production of single-stranded polynucleotide by dissociation of theelongation product from the above primer FA. These reactions are notshown since they are not thought to lead to the production of newtemplate polynucleotides.

Ultimately, all of the products shown in FIG. 2-(14) through FIG. 2-(18)function as starting substances for new complementary strand synthesisby becoming template polynucleotides in the present invention. Namely,they lead to the formation of structures shown as “recycling products”in FIG. 1. The polynucleotide amplification method based on the presentinvention continues in this manner.

A template polynucleotide of the present invention can be synthesized byusing as a starting material, a target nucleotide sequence comprised ina polynucleotide double-stranded state. A method for carrying outcomplementary strand synthesis by annealing a primer to a targetnucleotide sequence in a double-stranded state has been reported by theinventors (23rd Annual Meeting of the Japan Molecular Biology Society,Dec. 13-16, 2000, Kobe, Nagamine, K., Watanabe, K., Ohtsuka, K., Hase,T., and Notomi, T. (2001), Loop-mediated isothermal amplificationreaction using a non-denatured template, Clin. Chem. 47, 1742-1743).

In FIG. 1, a template polynucleotide is synthesized by using asingle-stranded target nucleotide sequence as a starting substance.However, if a method is applied in which a target nucleotide sequencecontained in a polynucleotide in a double-stranded state is used as thestarting substance, the step of denaturing to a single strand can beomitted, and the double-stranded polynucleotide can be used directly asa starting substance. A double-stranded polynucleotide of the presentinvention includes, for example, cDNA and genome DNA. In addition,various vectors into which these DNAs have been inserted can be alsoused as double-stranded polynucleotides of the present invention. On theother hand, single-stranded polynucleotides include those obtained bydenaturing double-stranded polynucleotides with heat, alkaline, or such,and those that originally exist as single-stranded polynucleotides suchas mRNAs. The double-stranded polynucleotides of the present inventionmay be purified or crude nucleic acids. Moreover, the method of thepresent invention is also applicable to nucleic acid in cells (in situ).In-situ genomic analysis can be achieved using a polynucleotide in cellsas template.

When a cDNA is used as a template in the present invention, the step ofcDNA synthesis and the method of polynucleotide synthesis according tothe present invention can be carried out under the same conditions. Whenfirst strand synthesis of cDNA is carried out using RNA as template, adouble-stranded polynucleotide of a DNA-RNA hybrid is formed. The methodfor synthesizing a polynucleotide can be conducted using thedouble-stranded polynucleotide as template of the present invention.When a DNA polymerase used in a method for synthesizing a polynucleotideof the present invention has a reverse transcriptase activity, thepolynucleotide synthesis can be achieved using a single enzyme under thesame conditions. For example, Bca DNA polymerase is a DNA polymerasehaving strand-displacing activity as well as reverse transcriptaseactivity. A method for synthesizing a polynucleotide according to thepresent invention can be also used after the formation of completedouble-stranded cDNA by the second strand synthesis.

When a template polynucleotide is synthesized from a target nucleotidesequence in a double-stranded polynucleotide, an arbitrary primer isfirst mixed with the double-stranded polynucleotide, and the mixture isincubated under conditions ensuring complementary strand synthesis usingthe primer as the starting point. Herein, the arbitrary primer is usedto place in condition for base pairing, the region to which the firstprimer will anneal to. Thus, the arbitrary primer is required to havethe ability to anneal to the complementary strand of the polynucleotidestrand in the target nucleotide sequence, to which the first primeranneals. Further, the strand extension in the complementary strandsynthesis using the arbitrary primer of the present invention as thereplication origin should proceed toward the region to which the firstprimer anneals. In other words, the primer can anneal to an arbitraryportion of the region, which region serves as the template in acomplementary strand synthesis using the first primer as a startingpoint. The arbitrary primer can be selected from arbitrary regions solong as it meets the criteria. For example, the second primer can bealso used as the arbitrary primer. The use of the outer primer is one ofthe preferred embodiments of the present invention, due to the reducednumber of necessary reaction components.

Thus, in the step a) of the reaction for synthesizing a templatepolynucleotide based on LAMP method, i.e. the step in which the firstprimer anneals to the target nucleotide sequence to start thecomplementary strand synthesis, base pairing with the first primer canbe ensured by displacing one of the two strands of the double-strandedpolynucleotide in complementary strand synthesis using the arbitraryprimer as a starting point. By choosing such a condition, the method forsynthesizing can be conducted without changing temperature. A conditionwhich ensures the annealing of the arbitrary primer and double-strandedpolynucleotide, and complementary strand synthesis using this primer asa starting point, is practically a condition where the followingmultiple steps can be achieved without changing the reaction condition:

i) annealing of the primer to a double-stranded polynucleotide template;and

ii) proceeding with complementary strand synthesis using the annealingprimer as the replication origin.

It was believed that a primer could anneal to a nucleic acid strand onlyif the region to which the primer anneals is single-stranded. Thus, whena double-stranded polynucleotide is used as a template, the nucleic acidhad to be subjected to a step that converts the nucleic acid into singlestrands by denaturation prior to the primer annealing. However, thereaction of complementary strand synthesis using a primer as a startingpoint can be achieved by incubating the template with the primer underconditions where the double strand is destabilized by whatever meanswithout completely converting the double stand into single strands. Thecondition under which the double-stranded nucleic acid is heated up tonearly the melting temperature (hereinafter abbreviated as Tm) can beexemplified as such a double strand-destabilizing condition.Alternatively, the addition of a Tm regulator is also effective.

In a method for synthesizing a polynucleotide of the present invention,a reaction comprising a series of steps is carried out in the presenceof a buffer giving a pH suitable for the enzyme reaction, salts requiredfor primer annealing and maintaining the catalytic activity of theenzyme, preservatives for the enzyme, and in addition if needed, amelting temperature (Tm) regulator, and such. The buffer with abuffering action in a range from neutral to weak alkaline pH, such asTris-HCl, is used in the present invention. The pH is adjusted dependingon the type of DNA polymerase used. Examples of salts to be added tomaintain the enzyme activity and to modify the melting temperature (Tm)of the polynucleotide include KCl, NaCl, MgCl₂, MgSO₄, (NH₄)₂SO₄, etc.Enzyme preservatives include bovine serum albumin and sugars.

Further, typical melting temperature (Tm) regulators include betaine,proline, dimethylsulfoxide (hereinafter abbreviated as DMSO), formamide,and trimethylamine N-oxide (hereinafter, abbreviated as TMANO). When amelting temperature (Tm) regulator is used, annealing of theabove-mentioned oligonucleotide can be regulated within a limitedtemperature range. Moreover, betaine (N,N,N-trimethylglycine) andtetraalkylammonium salts effectively contribute to the improvement ofthe efficiency of strand displacement due to its isostabilizing action.The addition of betaine at a concentration of about 0.2 to 3.0 M,preferably about 0.5 to 1.5 M to the reaction solution is expected toenhance the amplification of polynucleotides of the present invention.Since these melting temperature regulators decrease the meltingtemperature, a condition giving desired stringency and reactivity ischosen by considering reaction conditions such as salt concentration andreaction temperature.

Suitable temperature conditions for enzyme reactions can be readilychosen by utilizing a Tm regulator. Tm alters depending on the relationof the primer and target nucleotide sequence. Thus, it is preferable toadjust the amount of a Tm regulator so that the conditions that maintainenzyme activity are consistent with the incubation conditions that meetthe criteria of the present invention. Based on the disclosure of thepresent invention, those skilled in the art can readily choose properamounts of a Tm regulator to be added, depending on the primernucleotide sequence. For example, Tm can be determined based on thelength of the annealing nucleotide sequence and the GC content, saltconcentration, and concentration of the Tm regulator.

Annealing of a primer to a double-stranded polynucleotide under suchconditions is presumed to be unstable. However, complementary strandsynthesis proceeds using the unstable annealed primer as the replicationorigin when DNAs are incubated with a polymerase-displacing strand. Oncea complementary strand is synthesized, primer annealing becomes morestable over time. The DNA polymerases listed below catalyzecomplementary strand synthesis under conditions ensuring primerannealing to the double-stranded nucleic acid.

All primers used in the present invention can be chemically synthesized.The method for synthesizing a DNA is well known. Alternatively,naturally-occurring polynucleotides can be truncated and altered orlinked so as to comprise necessary sequences. Furthermore, all primersused in the present invention can be those that are artificiallymutagenized as well as those having a structure of a naturally-occurringDNA. A primer has to meet two requirements: (1) has to be able to formcomplementary base pairing with a target nucleotide sequence, and (2)provide an —OH group at the 3′-end of the base pair that serves as thecomplementary strand synthesis origin. Moreover, it is preferable thatthe primer can be a template for complementary strand synthesis. Thebackbone of the primer is not restricted to those composed ofphosphodiester bonds. For example, the primer may be a phosphothioate ormay comprise peptide nucleic acids based on peptide bindings. Further,the nucleotide may be any nucleotide, so long as it forms acomplementary base pair. In general, there are five types of naturallyoccurring nucleotides, namely A, C, T, G, and U; however, analogues suchas bromodeoxyuridine, for example, are also included. An oligonucleotideused in the present invention serves not only as a starting point forsynthesis, but preferably acts also as a template for complementarystrand synthesis.

The primer used in the present invention consists of nucleotides withappropriate lengths to enable base pairing with the complementary strandby maintaining required specificity under a given condition in varioustypes of polynucleotide synthesis reactions in the present invention.Specifically, a primer comprises 5 to 200 nucleotides, and morepreferably 10 to 50 nucleotides. The minimal length of a primerrecognized by known polymerases catalyzing sequence-dependentpolynucleotide synthesis is around 5 nucleotides. Thus, the length of anannealing portion should be longer than 5 nucleotides. In addition, toensure nucleotide-sequence specificity, a primer comprisesstochastically 10 nucleotides or more. On the other hand, an overly longnucleotide sequence is difficult to chemically synthesize. Thus, theabove-mentioned length of primers are exemplified as the preferredrange. The exemplified length of primers correspond only to the portionannealing to the complementary strand. For example, RA consists of atleast two regions, R2 and R1c. Thus, the above exemplified length ofprimers should be understood as a length corresponding to the length ofeach region constituting the primer.

The term “template” as used in the present invention refers to apolynucleotide that serves as a template in complementary strandsynthesis. Although a complementary strand having a nucleotide sequencethat is complementary to a template is a strand corresponding to thetemplate, the relationship between the two is merely relative.Specifically, a strand synthesized as a complementary strand has theability to function as a template. In other words, a complementarystrand can also serve as a template.

A DNA polymerase catalyzing the complementary strand synthesis reactionthat comprises strand displacement is used in the methods forsynthesizing or amplifying the polynucleotide the present invention. Thesame type of polymerases as those used for SDA and such can be used asDNA polymerases of the present invention. A specific polymerase thatsynthesizes complementary strands by displacing the double-strandedregion at the 5′ side, if any double-stranded region exists on thetemplate, during complementary strand synthesis using a primercomplementary to a region at the 3′-side of a certain nucleotidesequence as the synthesis origin, is known in the art. Thus, the 5′-sideof the template is the direction in which the reaction of complementarystrand synthesis proceeds. In the present invention, substrates requiredfor complementary strand synthesis are further added.

A DNA polymerase catalyzing a complementary strand synthesis reactionaccompanying strand displacement plays a central role in a method forsynthesizing a polynucleotide of the present invention. Such DNApolymerases include those listed below. In addition, various mutants ofthese enzymes can be used in the present invention, so long as they havethe activity of sequence-dependent complementary strand synthesis andthe strand displacing activity. Such mutants include truncated enzymeshaving only the structures with catalytic activity or mutant enzymeswhose catalytic activity, stability, or thermal stability has beenmodified by amino acid mutations, and such.

-   Bst DNA polymerase-   Bca(exo-) DNA polymerase-   Klenow fragment of DNA polymerase I-   Vent DNA polymerase-   Vent(Exo-) DNA polymerase (exonuclease activity-free Vent DNA    polymerase)-   DeepVent DNA polymerase-   DeepVent(Exo-) DNA polymerase (exonuclease activity-free DeepVent    DNA polymerase)-   Φ29 phage DNA polymerase-   MS-2 phage DNA polymerase-   Z-Taq DNA polymerase′ (Takara Shuzo)-   KOD DNA polymerase (TOYOBO)

Among these enzymes, Bst DNA polymerase and Bca(exo-) DNA polymerase areparticularly preferred, because they are enzymes with thermal stabilityto a certain degree and high catalytic activity. In the presentinvention, particularly when using a double-stranded polynucleotide as atemplate, the annealing of a primer and complementary strand synthesisare conducted under the same conditions. Since such reactions oftenrequire some heating, the use of thermostable enzymes is preferred. Thereaction can be achieved under a wide variety of conditions bythermostable enzymes.

For example, Vent(Exo-) DNA polymerase is a highly thermostable enzymethat has strand displacing activity. It has been reported that theaddition of a single strand-binding protein accelerates the reaction ofcomplementary strand synthesis by DNA polymerase which comprises stranddisplacement (Paul M. Lizardi et al., Nature Genetics 19, 225-232, July,1998). By applying the method to the present invention, acceleration ofcomplementary strand synthesis is expected by the addition of a singlestrand-binding protein. When Vent(Exo-) DNA polymerase is used, T4 gene32 is effective as the single strand-binding protein.

DNA polymerases lacking 3′-5′ exonuclease activity is known to have aphenomenon where the complementary strand synthesis is not terminatedeven when the reaction reaches the 5′-end of the template and synthesisgoes on until an extra nucleotide is added to the synthesized strand.Such a phenomenon is not preferable in the present invention, becausethe next complementary strand synthesis initiates from the synthesized3′-end complementary strand sequence. However, the nucleotide added tothe 3′-end by the DNA polymerase will be nucleotide “A” with a highprobability. Thus, a sequence for complementary strand synthesis shouldbe selected so as to initiate synthesis from the 3′-end from A to avoidproblems by the erroneous addition of a single-dATP nucleotide.Alternatively, even when the 3′-end protrudes during complementarystrand synthesis, it can be digested to a blunt end by a 3′→15′exonuclease activity. For example, the natural Vent DNA polymerase,which has such a activity, can be used in combination with Vent(Exo-)DNA polymerase to overcome the problem.

Unlike the DNA polymerases described above, DNA polymerases such as Taqpolymerase that are routinely used in PCR and such, exhibitsubstantially no strand displacement activity under usual conditions.However, such DNA polymerases can also be used for the presentinvention, when they are used under conditions ensuring stranddisplacement.

Moreover, the present invention relates to a polynucleotide detectionmethod based on the above polynucleotide amplification methods. Namely,the amount or presence of a target nucleotide sequence can be determinedby using the amount or presence of products according to the aboveamplification methods as indexes. Methods for detecting polynucleotidesare known. For example, an intercalator like ethidium bromide(abbreviated as EtBr) emits fluorescence by reacting withdouble-stranded DNA. The amount or presence of a product of anamplification reaction based on the present invention can be determinedby using this type of indexes.

The following method, for example, can be used to determine the amountof a polynucleotide based on a polynucleotide detection method of thepresent invention. To begin with, a method can be used that measures thetime required to impart a fixed signal. Since the amplification methodsof the present invention are volume-dependent reactions, the time untilthe reaction reaches a plateau is influenced by the initially presentamount of the polynucleotide serving as a template. Thus, provided otherreaction conditions are the same, the amount of a polynucleotide can beexpressed as a function of the time until the reaction reaches aplateau. In addition, the abundance of a polynucleotide can also beexpressed as a function of the amplification product amount formed in afixed reaction time.

In the LAMP method, an advanced amplification reaction takes place whenthe region to which the primer of a target nucleotide sequence is toanneal is in a suitable positional relationship, and its nucleotidesequence is as designed. In other words, the LAMP method is considerablyinhibited when the target nucleotide sequence is different from thepredicted nucleotide sequence in the region that must serve as astarting point for complementary strand synthesis. Complementary strandsynthesis in which a 3′-end that anneals to itself serves as thestarting point is particularly important. In the LAMP method, a 3′-endthat anneals to itself is equivalent to a 3′-end of a complementarystrand synthesized by using as a template, a nucleotide sequencearranged on the 5′-end of a primer. Thus, it is preferable to arrange anucleotide complementary to a mutated site to be detected on the 5′-endor its vicinity of a region (R1 or F1) arranged on the 5′-side of afirst primer (RA) and/or second primer (FA) Therefore, if designed sothat this important sequence corresponds to a mutation to be detected,the presence of mutations such as nucleotide deletions or insertions, orgenetic polymorphisms such as SNPs can be analyzed by observing theamplification reaction product according to the LAMP method.

More specifically, a nucleotide for which a mutation or polymorphism ispredicted is designed so as to be equivalent to the vicinity of the3′-end that serves as the starting point for complementary chainsynthesis (or the vicinity of the 5′-end when the complementary strandis the starting point). Namely, it is designed so that, when anynucleotide is different from the predicted nucleotide in a region towhich a primer or 3′-end complementary to itself anneals, complementarystrand synthesis using that primer as a starting point is impaired. Forexample, in the case where there is a mismatch within 10 nucleotidesfrom the 3′-end, particularly in the 2nd to 4th nucleotides countingfrom the 3′-end, and more preferably in the 2nd or 3rd nucleotidescounting from the 3′-end, which serves as a starting point forcomplementary strand synthesis, complementary strand synthesis issignificantly inhibited. Here, the predicted nucleotide sequence may bea wild type sequence or a mutated sequence. In the case where a mutatedsequence is predicted, complementary strand synthesis starts only whenthere has been a specific mutation. A polynucleotide complementarystrand synthesis reaction is significantly inhibited when there is amismatch in the 3′-end or its vicinity that serves as the starting pointfor complementary strand synthesis. Thus, when an amplification reactionis not inhibited, it can be judged that the target nucleotide sequenceis composed of the predicted nucleotide sequence. Conversely, when theamplification reaction is inhibited and a product is not formed to thesame degree as a control, it can be judged that the target nucleotidesequence differs from the predicted nucleotide sequence.

The LAMP method does not lead to an advanced amplification reactionunless the reaction is repeatedly carried out on the end structure ofthe initial reaction product. Thus, even if synthesis is carried outincorrectly, since complementary strand synthesis that composes theamplification reaction is always inhibited at each stage, advancedamplification does not occur while containing a mismatch. As a result, amismatch effectively represses the amplification reaction, andultimately leads to the obtaining of the correct result. In other words,a polynucleotide amplification reaction based on the LAMP method can besaid to have a more highly complete nucleotide sequence check mechanism.These characteristics offer advantages that are unlikely to be expectedwith methods such as the PCR method in which the amplification reactionis simply carried out in two regions. The present applicant has filed apatent application relating to a polymorphism and mutation detectionmethod based on the LAMP method (WO 01/34838).

The present invention provides a method for detecting a mutation in atarget nucleotide sequence by applying a loop primer of the presentinvention wherein, when a specific nucleotide in a target nucleotidesequence is not the predicted nucleotide, at least one complementarystrand reaction synthesis selected from complementary strand reactionsthat use the 3′-ends of an inner primer, loop primer, and the elongationproducts of these primers as a starting point is impaired, and whetheror not a target nucleotide sequence is the predicted nucleotide sequenceis detected by using a product of the above amplification reaction as anindex. By observing the production of an amplification reaction productformed based on the above complementary strand synthesis reactions, aspecific nucleotide can be judged as not being the predicted nucleotidewhen the production is inhibited compared to when the target nucleotidesequence is the predicted nucleotide sequence.

In order to detect whether or not a specific nucleotide in a targetsequence is the predicted nucleotide by applying the LAMP method basedon the present invention, when, for example, a complementary strandsynthesized by using the 5′-end of an inner primer as template startscomplementary strand synthesis by annealing to itself, it should bedesigned so that the complementary strand synthesis is controlled by theabove specific nucleotide. The present invention can also be designed sothat a complementary strand synthesis reaction that starts from the3′-end of an inner primer is regulated. However, in order to takeadvantage of the characteristics of the LAMP method in which annealingto a template nucleotide sequence is carried out repeatedly, it ispreferable to arrange a checking sequence on the 5′-side of the innerprimer. In the present invention, a checking sequence refers to anucleotide sequence that satisfies the following conditions of (a), (b),and (c).

-   (a) The checking sequence is arranged on the 5′-end of an inner    primer, and the 3′-end of the complementary strand synthesized by    using its nucleotide sequence as a template serves as a starting    point for complementary strand synthesis by annealing to a target    nucleotide sequence or its complementary strand.-   (b) In the case where the nucleotide to be checked is not the    predicted nucleotide, a mismatch occurs when the 3′-end of (a)    anneals to a target nucleotide sequence or its complementary strand.-   (c) The complementary strand synthesis of (a) is inhibited by the    mismatch that occurred in (b).

Namely, the present invention provides a method for detecting whether ornot a specific nucleotide in a target nucleotide sequence is thepredicted nucleotide by combining with a loop primer of the presentinvention using the following first primer and second primer as innerprimers, wherein at least either of the first primer or second primercomprises a checking sequence on its 5′-side.

A checking sequence refers to a nucleotide sequence in which, when anucleotide sequence that composes the above specific region is not thepredicted nucleotide sequence, a mismatch occurs when the 3′-end of thecomplementary strand synthesized using the checking sequence as templateis annealed to the target nucleotide sequence or its complementarystrand, and a complementary strand synthesis reaction that starts byusing the 3′-end as a starting point is inhibited by this mismatch.

First primer: The first primer (i) can provide at its 3′-end a startingpoint for complementary strand synthesis to a region that defines the3′-side of one of the strands that composes a target nucleotidesequence, and (ii) has on its 5′-side a nucleotide sequence that iscomplementary to an arbitrary region of the complementary strandsynthesis reaction product that uses this primer as a starting point.

Second primer: The second primer has (i) on its 3′-end a nucleotidesequence that is capable of providing a starting point for complementarystrand synthesis to a region that defines the 3′-side of a targetnucleotide sequence in an elongation product that uses the above firstprimer as a starting point, and (ii) on its 5′-side a nucleotidesequence that is complementary to an arbitrary region of thecomplementary strand synthesis reaction product that uses this primer asa starting point.

The location that provides a mismatch based on a difference in aspecific nucleotide by using a checking sequence is preferably, forexample, within 10 nucleotides from the 3′-end, particularly preferablythe 2nd to 4th nucleotides counting from the 3′-end, and more preferablythe 2nd or 3rd nucleotide counting from the 3′-end, which serves as astarting point for complementary strand synthesis. In the presentinvention, a mismatch at this location most effectively inhibitscomplementary strand synthesis. Here, the predicted nucleotide sequencemay be a wild type sequence or mutated sequence. The polynucleotidecomplementary strand synthesis reaction is significantly inhibited whena mismatch is present in the 3′-end or its vicinity that serves as astarting point for complementary strand synthesis.

Each of the above methods is for checking a specific nucleotide in atarget nucleotide sequence by placing a checking sequence on the 5′-sideof an inner primer. In the LAMP method, the 3′-end of a complementarystrand synthesized by using the nucleotide sequence arranged on the5′-side of the inner primer as template, is useful for checking aspecific nucleotide in a target nucleotide sequence by using the productof the complementary strand synthesis reaction as an index.

Moreover, the present invention provides a method for checking aspecific nucleotide in a target nucleotide sequence by using anucleotide sequence arranged on the 5′-side of not only an inner primer,but also a loop primer. Namely, the present invention provides a methodfor detecting whether or not a specific nucleotide in a targetnucleotide sequence is the predicted nucleotide by using the followingfirst loop primer and/or second loop primer as a loop primer along withthe above inner primer comprising a checking sequence. At this time,however, when the loop primer comprises a nucleotide sequencecomplementary to the previously mentioned arbitrary region arranged onthe 5′-side of the inner primer, or when the nucleotide sequencearranged on the 5′-side of the inner primer is a checking sequence, asequence in which the nucleotide for providing the above mismatch in thechecking sequence differs from the checking sequence placed on the5′-side of the loop primer. When the nucleotide sequence placed on the5′-side of the inner primer is a checking sequence, a sequence in whichthe nucleotide for providing the mismatch differs from the checkingsequence, may be different for the nucleotide only, or a plurality ofnucleotides including the nucleotide may be different.

First loop primer: Provides, between a region derived from a first innerprimer in an elongation product of the first inner primer and the abovearbitrary region with respect to the first inner primer, a startingpoint for complementary strand synthesis.

Second loop primer: Provides, between a region derived from a secondinner primer in an elongation product of the second inner primer, andthe above arbitrary region with respect to the second inner primer, astarting point for complementary strand synthesis.

A major characteristic of a specific nucleotide check mechanism based onthe LAMP method is that a complementary strand synthesis reaction thatuses a 3′-end annealed to a template or its complementary strand as astarting point occurs repeatedly. Due to this characteristic,differences in nucleotides in the template can be sensitively detectedby using the complementary strand synthesis reaction as an index.However, in the LAMP method as well, reactions occur that are notmediated by a checking mechanism. Since reactions not mediated by achecking mechanism yield products that are not produced as a result ofchecking the nucleotide sequence of the template, such reaction productshave the potential to impair judgment of results. Products that causethis reaction gradually accumulate in the reaction system during thecourse of repeating the amplification reaction according to the LAMPmethod.

In an analytical method using an amplification reaction as an index, animportant condition for a more sensitive detection is creating as alarge a difference as possible in the product amount and reaction rateof the amplification reaction between the case when the amplificationreaction proceeds and when the reaction is inhibited. However, ifnumerous reaction products are present that are formed unrelated to atemplate nucleotide, it becomes difficult to creating a large differencebetween the two. In other words, there is the risk of a loss ofsensitivity of the analytical method that uses the amount ofamplification product formed and its rate of formation as indexes.

In the present invention, the use of the 5′-side of a loop primer forchecking a specific nucleotide in a target nucleotide sequence has theeffect of repressing the products that have the potential to inhibitjudgment of such results. The following provides an explanation of thefunction of a nucleotide sequence arranged on the 5′-side of a loopprimer in the present invention.

The nucleotide sequence arranged on the 5′-side of the loop primerprovides a 3′-end that serves as the starting point for complementarystrand synthesis by annealing to itself in a complementary strandproduced by using the primer as template. At this time, complementarystrand synthesis that uses that 3′-end as a starting point is inhibitedin the case where a specific nucleotide is the predicted nucleotide. Thereason for this is as described below.

As was previously stated, in the method of the present invention, achecking sequence arranged on the 5′-side of an inner primer is designedso that, in the case where a specific nucleotide is not the predictednucleotide in the 3′-end of a complementary strand produced by using itas a template, complementary strand synthesis is inhibited by thatmismatch. In other words, the checking sequence of the inner primer issuch that a complementary strand synthesis reaction using as a startingpoint the 3′-end of a complementary strand used for a template, proceedswhen a specific nucleotide is the predicted nucleotide. In this method,products resulting from undesirable reactions that end up occurring whena specific nucleotide in a template nucleotide sequence is not thepredicted nucleotide, comprise nucleotide sequences that are displacedwith nucleotides that differ from the template in which a specificnucleotide is the predicted nucleotide and so forth. This product wasproduced as a result of passing through the checking mechanism providedby the 3′-end of the complementary strand synthesized by using thechecking sequence as a template for some reason. To facilitate thefollowing explanation, in a complementary strand synthesis reaction thatuses as a starting point the 3′-end of a complementary strand producedby using the 5′-side of an inner primer as a template, a product inwhich the above specific nucleotide has been displaced is tentativelyreferred to as a pseudo product. The formation of pseudo products andnew complementary strand synthesis reactions using formed pseudoproducts as templates should be repressed in the case of analyzing aspecific nucleotide.

A specific nucleotide is not the predicted nucleotide in nucleotidesequences that compose pseudo products. In the case of a loop primer,since the reaction proceeds by using a pseudo product as template, thecomplementary strand synthesis reaction that uses as a starting pointthe 3′-end of a complementary strand produced by using 5′-side of theloop primer as template, is impaired in the case when a specificnucleotide is not the predicted nucleotide. Ultimately, thecomplementary strand reaction from the 3′-end that uses a pseudo productas template, is impaired. In this manner, undesirable reactions can besuppressed by using a nucleotide sequence arranged on the 5′-side of theloop primer.

On the other hand, even when a specific nucleotide in a targetnucleotide sequence is the predicted nucleotide, a complementary strandsynthesis reaction that uses as a starting point the 3′-end of acomplementary strand produced by using a nucleotide sequence arranged onthe 5′-side of a loop primer as a template, can be inhibited. However,since a complementary strand synthesis reaction that uses the 3′-side ofthe loop primer as a starting point proceeds unrelated to the nucleotidesequence arranged on its 5′-side, the accelerating reaction effectitself of the loop primer on the LAMP reaction is not inhibited.Moreover, the amount of the above pseudo products produced is onlyslight in contrast to the large amount of product produced when aspecific nucleotide is the predicted nucleotide. What is more, theinhibiting effect of the reaction due to the above-mentioned mechanism,acts on pseudo products. Finally, the differences in the amount ofreaction products and production rate are thought to be further enlargeddepending on whether or not a specific nucleotide is the predictednucleotide. The fact that a nucleotide sequence arranged on the 5′-sideof a loop primer has the effect of enlarging the above differences isconfirmed also in the examples.

Mutations of nucleotides in a target nucleotide sequence can beidentified by using a checking mechanism for a specific nucleotide thatuses the above-mentioned inner primer and nucleotide sequence arrangedon the 5′-side of a loop primer. Thus, the present invention provides amethod for determining whether a specific nucleotide in a targetnucleotide sequence is the first nucleotide or the second nucleotide,comprising the step of mixing the following elements a) through d) andthen incubating under conditions that enable a complementary strandsynthesis reaction accompanying strand displacement, and the formationrate and/or formed amount of the amplification product is measured byany one of the primer sets in a) selected from the group consisting of:

a)

-   -   (1): first nucleotide inner primer pair and first nucleotide        loop primer pair    -   (2): first nucleotide inner primer pair and second nucleotide        loop primer pair    -   (3): second nucleotide inner primer pair and first nucleotide        loop primer pair, and    -   (4): second nucleotide inner primer pair and second nucleotide        loop primer pair;

wherein, the first nucleotide inner primer pair and the secondnucleotide inner primer pair are both primer pairs consisting of thenext first inner primer and second inner primer, and in the firstnucleotide primer pair, a complementary strand synthesis reaction usingas the starting point the 3′-end of the complementary strand synthesizedusing the 5′-sides of the first inner primer and second inner primer asa template is not inhibited when the specific nucleotide in the targetnucleotide sequence is the first nucleotide, but is inhibited when it isthe second nucleotide;

in the second nucleotide inner primer pair, a complementary strandsynthesis using as the starting point the 3′-end of a complementarystrand synthesized using the 5′-sides of the first inner primer andsecond inner primer as a template is not inhibited when the specificnucleotide in the target nucleotide sequence is the second nucleotide,but is inhibited when it is the first nucleotide;

the first inner primer has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of one of the strands that compose a targetnucleotide sequence, and (ii) on the 5′-side a nucleotide sequence thatis complementary to the arbitrary region of a complementary strandsynthesis reaction product that uses this inner primer as an startingpoint;

the second inner primer has (i) on its 3′-end a nucleotide sequence thatprovides a starting point for complementary strand synthesis to a regionthat defines the 3′-side of a target nucleotide sequence in anelongation product that uses the first inner primer as an startingpoint, and (ii) on the 5′-side a nucleotide sequence that iscomplementary to the arbitrary region of a complementary strandsynthesis reaction product that uses this inner primer as an startingpoint;

the first nucleotide loop primer pair and the second nucleotide loopprimer pair are both pairs consisting of the next first loop primer andsecond loop primer, and in the first nucleotide loop primer pair, acomplementary strand synthesis reaction using as the starting point the3′-end of the complementary strand synthesized using the 5′-sides of thefirst loop primer and second loop primer as template is not inhibitedwhen the specific nucleotide in the target nucleotide sequence is thefirst nucleotide, but is inhibited when it is the second nucleotide;

in the second nucleotide inner primer pair, a complementary strandsynthesis reaction using as the starting point the 3′-end of acomplementary strand synthesized using the 5′-sides of the first loopprimer and second loop primer as a template is not inhibited when thespecific nucleotide in the target nucleotide sequence is the secondnucleotide, but is inhibited when it is the first nucleotide;

the first loop primer provides, between a region derived from the firstinner primer in an elongation product of the first inner primer and thearbitrary region with respect to the first inner primer, a startingpoint for complementary strand synthesis, and

the second loop primer provides, between a region derived from thesecond inner primer in an elongation product of the second inner primerand the arbitrary region with respect to the second inner primer, astarting point for complementary strand synthesis;

b) a DNA polymerase catalyzing complementary strand synthesisaccompanying strand displacement;

c) a substrate for complementary strand synthesis; and

d) a test polynucleotide comprising a target nucleotide sequence.

In the explanations of the nucleotide sequences of each of the aboveprimers, inhibition of complementary strand synthesis refers to theoccurrence of a mismatch when a specific nucleotide is a certainnucleotide and the impairment of complementary strand synthesis causedby that mismatch when the 3′-end of a complementary strand producedusing a nucleotide sequence arranged on the 5′-side of each primer as atemplate anneals to a region containing a specific nucleotide in thesame manner as the checking sequence in an inner primer as previouslydescribed. The method for identifying a specific nucleotide of thepresent invention is useful for identifying nucleotide mutations andpolymorphisms. For example, the present invention can be used todetermine whether or not a certain nucleotide is the wild type ormutant. In this case, the method of the present invention can be carriedout by designing each of the above primers by using either a firstnucleotide or second nucleotide as a wild type and using the other as amutant. As a result, the nucleotide can be identified to be of the wildtype or mutant based on the combinations of primer sets when theproduction rate of the complementary strand synthesis reaction productreaches a maximum and a minimum. More specifically, the production ratesof amplification products when a specific nucleotide is of the wild typeand when a specific nucleotide is of the mutant, with respect to thefollowing four primer sets of (1) through (4), are the followingcombinations.

When the target nucleotide sequence is the wild type: (1) is maximum and(3) is minimum

When the target nucleotide sequence is a mutant: (4) is maximum and (2)is minimum

-   (1): Wild type inner primer pair and wild type loop primer pair-   (2): Wild type inner primer pair and mutant loop primer pair-   (3): Mutant inner primer pair and wild type loop primer pair-   (4): Mutant inner primer pair and mutant loop primer pair

This method is none other than a method for double checking a specificnucleotide. In this manner, the reliability of analysis results of aspecific nucleotide in a target nucleotide sequence can be improved byusing primer pairs composed of a plurality of primers. In methods foridentifying a specific nucleotide based on a known nucleic acidamplification reaction like PCR, there is no known method for enhancingthe reliability of analysis results in this manner.

The various types of reagents required for the polynucleotide synthesismethod according to the present invention, the amplification method thatuses this synthesis method, or method for detecting mutations in atarget nucleotide sequence that uses this amplification method, may beprovided by prepackaging in a kit. More specifically, a kit is providedfor carrying out the present invention that is composed of reagentsinvolving various oligonucleotides required for use as a first primer,second primer, and a loop primer, dNTPs to serve as the substrate ofcomplementary strand synthesis, DNA polymerase for carrying out stranddisplacement type complementary strand synthesis, and buffers forproviding suitable conditions for enzyme reactions. An outer primer mayalso be combined with the kit of the present invention. The combinationof outer primer allows isothermal reactions.

As already described, the polynucleotide amplification reactions of thepresent invention make it possible to detect target nucleotidesequences. Thus, a polynucleotide amplification reaction kit accordingto the present invention can be used as a kit for detecting a targetnucleotide sequence or as a kit for detecting mutations in a targetnucleotide sequence.

Namely, the present invention relates to kits for detecting a targetnucleotide sequence that contains the constituent elements describedabove. Moreover, a kit for detecting mutations in a target nucleotidesequence can be provided by using each of the primers that compose a kitfor detecting a target nucleotide sequence of the present inventionprimers for detecting mutations. In addition to an outer primer,reagents required for detecting synthesis reaction products may becombined with the kit according to the present invention as necessary.

Especially, in a preferable embodiment of the present invention, thereaction can be started simply by adding a sample, by providing areaction container containing reagents required for a single reaction,since it is not necessary to add reagents during the course of thereaction. If a system is provided that allows detection of the reactionproduct to be carried out directly in the reaction container by using aluminescent signal or fluorescent signal, the opening of the containerafter the reaction can be completely eliminated. This is highlydesirable in terms of preventing contamination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the principle of the polynucleotide amplificationreaction using the present invention. FA: Primer FA for LAMP method; RA:primer RA for LAMP method; loop F: Loop primer F; loop R: Loop primer R.

FIG. 2 shows the reaction principle of the polynucleotide amplificationmethod using the invention. Abbreviations used in the figure are thesame as in FIG. 1.

FIG. 3 shows the preferable positional relationship between loop primersand the sequence of the template polynucleotide.

FIG. 4 shows the positional relationship between the templatepolynucleotide sequence used in the Examples (SEQ ID NO: 1) and thenucleotide sequences of respective primers. The open-faced nucleotidesshow the annealing site for the loop primers and the arrows indicate thesynthesis direction of the complementary strand.

FIG. 5 shows the accelerating effect of loop primers on thepolynucleotide amplification reaction. The X-axis indicates the reactiontime and the Y-axis indicates the change in fluorescence intensity(ΔRn). Loop F/R: loop primer R and loop primer F were used. Loop cF/cR:loop cF and loop cR consisting of the complementary sequences of loopprimers F and R were used. −/−: loop primers were not added.

FIG. 6 shows the result of evaluating the amount of added template DNAand the amplified products. The X-axis indicates the reaction time andthe Y-axis indicates the change in fluorescence intensity (ΔRn).

FIG. 7 shows the concentration dependency of loop primers. The X-axisindicates the reaction time and the Y-axis indicates the change influorescence intensity (ΔRn).

FIG. 8 shows the effect of a 3′-aminated loop primer. The X-axisindicates the reaction time and the Y-axis indicates the change influorescence intensity (ΔRn). Loop primer: a Loop primer with anunmodified 3′ end was used. 3′ N-loop primer: Loop primer with anaminated 3′ end was used. −/−: a loop primer was not added.

FIG. 9 shows the effect of the presence or absence of outer primers onpolynucleotide amplification. The X-axis indicates the reaction time andthe Y-axis indicates the change in fluorescence intensity (ΔRn). Outerprimer (+): presence of outer primer. Outer primer (−): absence of outerprimer. Negative Control: template cDNA not added.

FIG. 10 shows the result of evaluating primer length. The X-axisindicates the reaction time and the Y-axis indicates the change influorescence intensity (ΔRn). F/R: loop primers R and F were used.F−1/R−1: loop primers F and R with 1 nucleotide less at the 5′end wereused. F−2/R−2: loop primers F and R with 2 less nucleotides at the 5′endwere used.

FIG. 11 shows the result of evaluating loop primer design site. TheX-axis indicates the reaction time and the Y-axis indicates the changein fluorescence intensity (ΔRn). F3/R4: loop primers F3 and R4 that donot overlap with the F2 and R2 regions, respectively, were used. F5/R5:loop primers F5 and R5 overlapping 3 nucleotides with the F2 and R2region, respectively, were used. F6/R6: loop primers F6 and R6overlapping 3 nucleotides with the F1 and R1 regions, respectively, wereused. F7/R7: loop primers F7 and R7 overlapping 10 nucleotides with theF1 and R1 regions, respectively, were used. F8/R8: loop primers F8 andR8 completely overlapping with the F1 and R1 regions, respectively, wereused. −/−: loop primers were not added.

FIG. 12 shows the result of SRY gene amplification according to thepresent invention. The X-axis indicates the reaction time and the Y-axisindicates the change in fluorescence intensity (ΔRn). Loop primer (+):presence of Loop primer. Loop primer (−): absence of loop primer.Negative Control: template DNA not added.

FIG. 13 shows the positional relationship between loop primer added tothe 5′ end and the template nucleotide sequence. “c” means complementarysequence.

FIG. 14 shows the accelerating effect of the loop primer added to the 5′end. The X-axis indicates the reaction time and the Y-axis indicates thechange in fluorescence intensity (ΔRn).

FIG. 15 shows the evaluation of SNP recognition at the 5′ end of a loopprimer. The X-axis indicates the reaction time and the Y-axis indicatesthe fluorescence intensity (ΔRn).

FIG. 16 shows the evaluation of the SNP recognition at the 5′ end of aninner primer and loop primer. The X-axis indicates the reaction time andthe Y-axis indicates the change in fluorescence intensity (ΔRn).

FIG. 17 illustrates the reaction principle of the SNP detection methodused in the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below with referenceto examples.

EXAMPLE 1 Accelerating Effect of Loop Primers on PolynucleotideAmplification

1. Design of the Loop Primers

To accelerate polynucleotide amplification by the LAMP method, theeffect of adding primers was tested. The design site of the loop primerswas set so as not to be within the R2 or F2 regions of loop structuresformed during the LAMP reaction. The direction of the primers was sameas R1 or F1. (Hereafter they will be described as loop primers). Thedirection of R1 or F1 indicates the same direction in which the templatepolynucleotides anneal to themselves to synthesize a complementarystrand. First, the following primers for the target sequence (SEQ IDNO: 1) were individually designed according to the known LAMP method (WO00/28082). The positional relationships between the templatepolynucleotide sequence (SEQ ID NO: 1) and each primer sequence areshown in FIG. 4. The open-faced nucleotides show the site to which theloop prier anneals and the arrow indicates the direction ofcomplementary strand synthesis.

-   Inner primer RA (Inner F, SEQ ID NO: 2),-   Inner primer FA (Inner R, SEQ ID NO: 3),-   Outer primer F (Outer F, SEQ ID NO: 4), and-   Outer primer R (Outer R, SEQ ID NO: 5).

The following primers were designed to be the same direction as R1 andF1 that are defined by the above primers.

-   Loop primer F (Loop F, SEQ ID NO: 6) and-   Loop primer R (Loop R, SEQ ID NO: 8).

All primers were synthesized by the known LAMP method.

By using loop primers, in addition to the reactions of the known LAMPmethod, one can expect another amplification reaction to occur, which isexpected to accelerate the amplification reaction (FIGS. 1 and 2).

2. Effect of Loop Primers

The LAMP reaction was carried out according to the reaction conditionsdescribed below using λDNA (1×10⁵ molecules) as template.Non-heat-denatured λDNA was prepared. The reaction was carried out at65° C. using the sequence detection system ABI PRISM 7700 (Perkin-ElmerBiosystems) and the transition of reaction was periodically monitored.

Reaction composition (in 25 μl)

-   20 mM Tris-HCl pH 8.8-   10 mM KCl-   10 MM (NH₄)₂SO₄-   4 mM MgSO₄-   1-M Betaine-   0.1% Triton X-100-   0.4 mM dNTP-   8 U Bst DNA Polymerase (NEW ENGLAND BioLabs)-   0.25 μg/ml EtBr    Primers:    -   1600 nM Inner F    -   1600 nM Inner R    -   400 nM Outer F    -   400 nM Outer R

SEQ ID NO: 1 is the target nucleotide sequence (λDNA 1) of λDNA. Loopprimers were simultaneously added to the final concentration of 400 nM.Consequently, a reaction accelerating effect was observed in thereaction system to which loop primers were added. On the other hand,primers consisting of complementary strands of loop primers (Loop cF/SEQID NO: 7 and Loop cR/SEQ ID NO: 9), had no effect (FIG. 5).

Various concentrations of λDNA (1×10², 1×10³, 1×10⁵ molecules) were usedas templates in the presence of loop primers to evaluate the detectionlimit. As a result, reproducible results were obtained when up to 1×10³molecules of the templates were used (FIG. 6). In the absence of loopprimers, even 1×10⁵ molecules of template DNAs gave variable results(data not shown), indicating that amplification of a low amount oftemplate can be accomplished by using loop primers.

2. Concentration Dependency of Loop Primers

Loop primers at final concentrations of 200, 400, 800, 1200, 1600, and2000 nM were added to the LAMP reaction system to test the effect ofchanging loop primer concentration on the LAMP reaction. Consequently,as loop primer concentration increased, acceleration of the LAMPreaction was observed (FIG. 7). A final concentration of 800 nM loopprimers was used in the examples hereafter.

3. Effect of 3′ Modified Loop Primers on the LAMP Reaction

To inhibit the extension reaction from a loop primer, a loop primeraminated at the 3′ end was prepared. The aminated loop primer was addedto the LAMP reaction at a final concentration of 800 nM to test theaccelerating effect. The result showed that in the presence of theaminated loop primer, no acceleration of reaction was observed. Thisindicated that the extension reaction from the loop primer is involvedin the acceleration of the LAMP reaction (FIG. 8).

4. Effect of Outer Primers

The effect of the absence or presence of outer primers on LAMP reactionwas tested in the presence of loop primers. In the absence of outerprimers, there was a 10-minute reaction delay (FIG. 9). This resultindicated that the combination of outer primers with loop primers wasalso advantageous in further accelerating the reaction.

5. Effect of Loop Primer Tm

The effect of loop primer Tm was tested by shortening the 5′ end ofprimers. Loop primers used in the experiments are as follows:

-   Loop primer F−1 lacking one nucleotide (Loop F−1, SEQ ID NO: 10);-   Loop primer R−1 lacking one nucleotide (Loop R−1, SEQ ID NO: 12);-   Loop primer F−2 lacking two nucleotides (Loop F−2, SEQ ID NO: 11);    and-   Loop primer R−2 lacking two nucleotides (Loop R−2, SEQ ID NO: 13).

As a result of using these primers, the rate of reaction slowed downwhen primers lacking one nucleotide were used, and the rate was furtherreduced when primers lacking two nucleotides were used (FIG. 10). It wassuggested that the reduction of reaction rate was due to the decrease ofloop primer Tm that made the reaction more difficult at 65° C.

6. Effect of Loop Primer Design Site

To evaluate the loop primer design site, new primers were designed in adifferent region of λDNA. The sequence of the target nucleotide sequence(λDNA2) is shown as SEQ ID NO: 14. Following LAMP primers were designedto amplify the target nucleotide sequence.

-   Inner primer RA (Inner F, SEQ ID NO: 15),-   Inner primer FA (Inner R, SEQ ID NO: 16),-   Outer primer F (Outer F, SEQ ID NO: 17), and-   Outer primer R (Outer R, SEQ ID NO: 18).

Then the following primers were designed to be the same direction as R1and F1 that are defined by above primers.

-   Loop primer F3: No overlapping with the R2 region (Loop F3, SEQ ID    NO: 19)-   Loop primer R4: No overlapping with the F2 region (Loop R4, SEQ ID    NO: 20)-   Loop primer F5: 3 nucleotides overlapping with the R2 region (Loop    F5, SEQ ID NO: 21)-   Loop primer R5: 3 nucleotides overlapping with the F2 region (Loop    R5, SEQ ID NO: 22)-   Loop primer F6: 3 nucleotides overlapping with the R1 region (Loop    F6, SEQ ID NO: 23)-   Loop primer R6: 3 nucleotides overlapping with the F1 region (Loop    R6, SEQ ID NO: 24)-   Loop primer F7: 10 nucleotides overlapping with the R1 region (Loop    F7, SEQ ID NO: 25)-   Loop primer R7: 10 nucleotides overlapping with the F1 region (Loop    R7, SEQ ID NO: 26)-   Loop primer F8: completely overlapping with the R1 region (Loop F8,    SEQ ID NO: 27)-   Loop primer R8: completely overlapping with the F1 region (Loop R8,    SEQ ID NO: 28)

The presence or absence of overlappings with the R2 and F2 regions didnot affect the reaction rate, and there was no difference in reactionrate among these loop primers. The primers in which 3 nucleotidesoverlapped with the R1 (or F1) region (loop F6/loop R6) were also used,but did not affect the reaction rate (FIG. 11).

Moreover, primers having a 10-nucleotide overlap in the R1 (or F1)region (loop F7/loop R7) were prepared. Although, the reaction rateslowed down with this setting compared to when there were 3 or lessoverlappings, an accelerating effect was still observed (FIG. 11). Forprimers that completely overlapped with the R1 (or F1) region (loopF8/loop R8), an accelerating effect was observed compared to thereaction without the loop primers (FIG. 11). The reasons for thereduction of the reaction rate may be the competitive inhibition in theR1 (or F1) region that made it more difficult to form the dumbbell-likestructure (FIG. 3), and the fact that primers had to anneal to the R1region that became double-stranded.

EXAMPLE 2 SRY Gene Amplification

The effect of loop primers on the LAMP reaction using human genome astemplate was tested. The SRY gene mapped on the Y chromosome wasselected as a target gene. The target nucleotide sequence is shown inSEQ ID NO: 29. To amplify the target nucleotide sequence, the followingLAMP were designed.

-   Inner primer RA (Inner F, SEQ ID NO: 30)-   Inner primer FA (Inner R, SEQ ID NO: 31)-   Outer primer F (Outer F, SEQ ID NO: 32), and-   Outer primer R (Outer R, SEQ ID NO: 33).

Then, the following loop primers were designed to be the same directionas R1 and F1 that were defined by the above primers.

-   Loop primer F (Loop F, SEQ ID NO: 34) and-   Loop primer R (Loop R, SEQ ID NO: 35).

The LAMP reaction was carried out using 100 ng of human genome.Consequently, it was confirmed that the reaction was accelerated whenthe loop primers were added (FIG. 12). That is, from this experiment,the accelerating effect of loop primers on the LAMP reaction wasconfirmed even when the human genome was used as template. It wasindicated that rapid genome analysis such as the detection of SNPs couldbe done based on the present invention.

EXAMPLE 3 Loop Primers with a Nucleotide Sequence Added to their 5′ Side

The acceleration of reaction rate was confirmed when a nucleotidesequence complementary to an arbitrary region of the complementarystrand synthesized from the 3′ end of a loop primer was added to the 5′side of the loop primer. The template used in this experiment was thesame λDNA (λDNA2) as described in Example 1. The reaction conditionswere also the same as in Example 1. First, primers F9-F15 were designedas shown in FIG. 13. At the 5′ side of the loop primers, variousnucleotide sequences were added. The following is a list of nucleotidesequences that were added to the 5′ side.

Loop primer Sequence at 5′ side Sequence at 3′ side F9 inner F1 F3 F10F1 F F11 F1 Fc F12 F F1 F14 inner F1c F2 F15 inner F1 F1

The positional relationship of each nucleotide sequence constituting theabove mentioned loop primers relative to the template nucleotidesequence are as shown in FIG. 13. Each nucleotide sequence is brieflydescribed below. Nucleotide sequence with a “c” means a complementarysequence.

The following explanation describes each regions of the loop thatinclude the nucleotide sequence of inner primer FA. However, in theexperiment, the same loop primers were also designed for loops thatinclude the inner primer RA sequence, and used. The primer sequencesactually used in the experiment are described at the end.

-   Inner F1: complementary nucleotide sequence of F1c located at the 5′    side of inner primer FA.-   F1: Region adjacent to the 5′ side of inner F1 in the loop.-   F2: Region partly overlapping with F1 in the loop and its 3′ end    located closer towards the 5′ side than F1.-   F3: Region partly overlapping with F1 in the loop and its 3′ end    located closer towards 5′ side than F2.-   F: Region adjacent region where inner primer F2c anneals in the    loop.

The LAMP reaction was carried out using these loop primers, and it wasshown that the reaction was accelerated compared to the reaction withoutloop primers.

In the previous experiment, it was demonstrated that the loop primerthat hybridizes to the loop to which the inner primer binds (loopCF/CR), does not have an effect on this reaction (FIG. 5). In thisexperiment, as shown in FIG. 14, the accelerating effect of the LAMPreaction was observed with the primer combination of F11/R11. At the 5′side of F11 and R11, primers have structures having nucleotide sequencescomplementary to arbitrary regions of the complementary strand extendedfrom their 3′ ends. This structure is the so-called inner primerstructure. Therefore, it was thought that the complementary strand thatwas synthesized using the loop primer as template, has a structure thatcauses the next extension reaction to occur using itself as template.

The following are the primer sequences used in this experiment:

F9/ CGTGAGCAATGGGTATATGCAAATGGAACTCCGGGTGCTATCAG/ SEQ ID NO:36 R9/ATGTCCTTGTCGATATAGGGATGAATGACCTTTCTCTCCCATATTGCAGTCG/ SEQ ID NO:37 F10/TTCGTTTCCGGAACTCCGGGTTGAATGCCCGGCGAACTGGAG/ SEQ ID NO:38 R10/TCGCTTGGTGTACCTCATCTACTGCGGCAGTCGCGGCACGATGGAACTA/ SEQ ID NO:39 F11/TTCGTTTCCGGAACTCCGGGTCTCCAGTTCGCCGGGCATTCA/ SEQ ID NO:40 R11/TCGCTTGGTGTACCTCATCTACTGCGTAGTTCCATCGTGCCGCGACTGC/ SEQ ID NO:41 F12/CTCCAGTTCGCCGGGCATTCATTCGTTTCCGGAACTCCGGGT/ SEQ ID NO:42 R12/TAGTTCCATCGTGCCGCGACTGCTCGCTTGGTGTACCTCATCTACTGCG/ SEQ ID NO:43 F13/TCCAGTTCGCCGGGCATTGTTTCCGGAACTCCGGGT/ SEQ ID NO:44 R13/TAGTTCCATCGTGCCGCGACTTCGCTTGGTGTACCTCATCTACTG/ SEQ ID NO:45 F14/ATTTGCATATACCCATTGCTCACGGGAACTCCGGGTGCTATCAG/ SEQ ID NO:46 R14/TTCATCCCTATATCGACAAGGACATTGACCTTTCTCTCCCATATTGCAGTCG/ SEQ ID NO:47 F15/CGTGAGCAATGGGTATATGCAAATTTCGTTTCCGGAACTCCGGGT/ SEQ ID NO:48 R15/ATGTCCTTGTCGATATAGGGATGAATCGCTTGGTGTACCTCATCTACTGCG/ SEQ ID NO:49

EXAMPLE 4 Detection of a Mutation with a Nucleotide Sequence Placed atthe 5′ Side of a Loop Primer

By utilizing a nucleotide sequence placed at the 5′ side of loop primersof the present invention, it was confirmed that when a specificnucleotide in the target nucleotide sequence was not the predicted one,the polynucleotide amplification method according to the presentinvention was inhibited. Therefore, by monitoring the presence orabsence of a reaction inhibition, one can detect a mutation of aspecific nucleotide.

At the 5′ side of a loop primer used for this experiment, the nucleotidesequence same as the one placed in the 5′ side of the inner primer wasplaced. That is, in the 5′ side of loop primer F, a nucleotide sequenceof the 5′ side of inner primer FA was placed. The 5′ side of loop primerR was designed to have the nucleotide sequence of the 5′ side from theinner primer RA. Moreover, in the 5′ end of each primer, primerscomprising nucleotides complementary to a mutant sequence (MT) andwild-type sequence (WT) were prepared, and for all combinations ofprimers, the amplification reactions based on the present invention wereperformed. When an amplification reaction based on the present inventionis conducted using these primers, a complementary strand is synthesizedusing each loop primer as template. Then when the complementary strandanneals to itself to start complementary strand synthesis, a specificnucleotide can be distinguished. In other words, the 3′ end of acomplementary strand synthesized using a primer (MT) comprisingnucleotides complementary to the mutant as template, can be used as astarting point for complementary strand synthesis when the specificnucleotide was a mutant. For example, if the 82^(nd) nucleotide of SEQID NO: 50 was T instead of A, the MT primer sequence was designed so asto initiate the expected amplification reaction. Also, the WT primercomprises nucleotides complementary to the wild-type nucleotides.Therefore, when it is utilized as a template to synthesize acomplementary strand, the 3′ end can act as a starting point forcomplementary strand synthesis for a wild-type nucleotide. That is, theWT primer can be referred to as a SNP recognition primer.

As template, a 106-mer polynucleotide comprising a λDNA-derivednucleotide sequence designated as SEQ ID NO: 50 was used. The reactionconditions were as follows:

Reaction composition (in 25 μL)   20 mM Tris-HCl pH 8.8   10 mM KCl   10mM (NH₄)₂SO₄   4 mM MgSO₄  0.8 M Betaine  0.1% Triton X-100  0.4 mM dNTP  8 U Bst DNA Polymerase (NEW ENGLAND BioLabs) 0.25 μg/ml EtBr Primers:800 nM Inner F 400 nM Loop F 800 nM Inner R 400 nM Loop R 200 nM Outer F200 nM Outer R

Template DNA and each primer sequence used in the experiment aredescribed below:

λDNA 35′-GCTCACTGTTCAGGCCGGAGCCACAGACCGCCGTTGAATGGGCGGATGCTAATTACTATCTCC (SEQID NO:50)CGAAAGAATCCGCATACCAGGAAGGGCGCTGGGAAACACTGCCCTTTCAGCGGGCCATCATGAATGCGATGGGCAGCGACTACATCCG-3′ InnerF_WTTGGTATGCGGATTCTTTCGGGAGGCTCACTGTTCAGGCCGGAG (SEQ ID NO:51) InnerR_WTAGGAAGGGCGCTGGGAAACACTCGGATGTAGTCGCTGCCCATC (SEQ ID NO:52) InnerF_MTAGGTATGCGGATTCTTTCGGGAGGCTCACTGTTCAGGCCGGAG (SEQ ID NO:53) InnerR_MTTGGAAGGGCGCTGGGAAACACTGGGATGTAGTCGCTGCCCATC (SEQ ID NO:54) OuterFAACAGGCTGCGGCATTTTGTC (SEQ ID NO:55) OuterR GGCAGACTTCACCACATTCACCTC(SEQ ID NO:56) LoopF GAGATAGTAATTAGCATCCGCC (SEQ ID NO:57) LoopRCACTGCCCTTTCAGCGGGCCAT (SEQ ID NO:58) LoopF_WTTGGTATGCGGATTCTTTCGGGAGGAGATAGTAATTAGCATCCGCC (SEQ ID NO:59) LoopR_WTAGGAAGGGCGCTGGGAAACACTCACTGCCCTTTCAGCGGGCCAT (SEQ ID NO:60) LoopF_MTAGGTATGCGGATTCTTTCGGGAGGAGATAGTAATTAGCATCCGCC (SEQ ID NO:61) LoopR_MTTGGAAGGGCGCTGGGAAACACTCACTGCCCTTTCAGCGGGCCAT (SEQ ID NO:62)

A Non-heat denatured template polynucleotide (λDNA, 10⁶ molecules) wasprepared. The reaction was carried out at 66° C. and using ABI PRISM7700 (Perkin-Elmer Biosystems), to periodically observe the transitionof the amplification.

The result of the reaction is shown in FIGS. 15 and 16. First, the loopprimers with the F1 (or R1) region added to the 5′ side were used toexamine if their 5′ ends were able to detect SNPs (FIG. 15). For theinner primer, a sequence that recognizes an SNP (WT) was used. Theresult demonstrated that the increase in the signal when using a loopprimer (MT loop) that cannot recognize an SNP at the 5′ end was slowercompared to that when using a primer that can recognize the SNP (WTloop). That is, this indicated that by changing the 5′ end of the aboveprimers to different nucleotides, an SNP could be detected based on thedifference in the reaction rate.

Next, the LAMP reaction was carried out using inner primers thatrecognize SNPs. Similar to Example 1, the inventor used loop primers, inparticular those with no additional specific sequences at their 5′sides, and only nucleotide sequences at the 5′ sides were changed. Theresult demonstrated that even when combining with a loop primer, asdescribed in the WO 01/34838 by the present applicant, utilization ofthe nucleotide sequences located at the 5′ side of inner primers enabledthe detection of a one-nucleotide difference (FIGS. 16-1 and 2).

In order to make the difference between FIGS. 16-1 and 2 larger, theabove-mentioned loop primer (WT loop) was used to conduct the LAMPreaction. Consequently, as shown in FIG. 16-4, the signal increased muchslower than in FIG. 16-2. The difference in reaction rates between wildtype and mutant could be clearly identified by using a loop primer witha nucleotide sequence added to the 5′ side, compared to when using onlythe inner primer.

When a mutant is detected based on the LAMP method, a smaller differencein signals can be seen between the wild type and mutant forms. One ofthe mechanisms behind this reduction of signal difference is thought tobe as follows. For instance, when the wild type is used as a template,it is assumed that the reaction proceeds using the inner primer (Mtinner) for detecting a mutation. When, this reaction product functionsas a template, the MT inner primer anneals to a portion produced usingthe primer sequence as template, and complementary strand synthesis isinitiated. As a result, a nucleotide substitution occurs at the SNPposition that should have been recognized, and the mutated template isamplified.

In this example, a mechanism to check for a nucleotide substitution inthe template was accomplished by using a loop primer capable of checkinga specific nucleotide sequence at the 5′ side. The principal of thischecking mechanism is shown in FIG. 17. Using this method, anunfavorable amplification reaction is repressed based on the mechanismas described above, resulting in a clear difference in signal as shownin FIG. 16-4.

INDUSTRIAL APPLICABILITY

The present invention provides a rapid polynucleotide synthesis method.The polynucleotide synthesis method of the present invention can beconducted by using template polynucleotides that can form loopstructures, and a plurality of primers that can bind to the loops toinitiate complementary strand synthesis. A template polynucleotide thatforms a loop structure can be synthesized easily by known methods suchas the LAMP method. By the application of the LAMP method, all reactionsinvolved can be performed at the same temperature. Therefore, in thepresent invention, by applying the LAMP method, all of the reactions canbe done at the same temperature. Moreover, the present inventionmaintains high specificity and a large amount of polynucleotides can beproduced in a short time.

Theoretically, the LAMP method rarely produces non-specific products.When the present invention is applied to the LAMP method, a loop primercan be designed in such a way that that it anneals to a specificreaction product. In this case, polynucleotides are synthesized rapidlyonly when appropriate reaction products are produced. Applying thepresent invention to the LAMP method makes it more difficult fornon-specific reactions to occur. Thus, the present invention enablesimprovement of not only reaction efficiency, but also reactionspecificity of the LAMP method.

The method of the present invention can be conducted simply by adding atleast one loop primer to the primers necessary for the LAMP method.Therefore, the method of the present invention can be referred to as aflexible method with a potential for wide use. A reagent for practicingthe present invention can be prepared by just adding a loop primer tothe reagents for the LAMP method. Thus, a reagent kit can be easilyproduced.

The LAMP method is a polynucleotide amplification method that is highlyspecific and easy to operate. The present invention provides all theadvantages of the LAMP method, and moreover, greatly increases thecomplementary strand synthesis reaction rate.

There is a growing need for analyzing SNPs and gene function, as well asgene diagnosis based on the results of those analyses. Thus, thedevelopment of a technology that enables gene nucleotide sequences to beanalyzed more accurately and rapidly is becoming an important issue, notonly in terms of rapidly carrying out functional analysis, but also withrespect to practical application of the results of gene functionanalysis in actual clinical settings.

In fact, clinical trials of pharmaceutical agents developed based on SNPanalyses have already commenced. When using such pharmaceutical agentsthat have passed the clinical trials, a method for simple and rapid SNPanalysis of patient genomes at hospital bedsides will be essential. Thepresent invention accomplished a useful gene analysis technique that canmeet such a high need.

1. A method for synthesizing a polynucleotide comprising the steps ofmixing the following elements 1 to 5, and incubating under conditionsthat allow template-dependent complementary strand synthesis using theDNA polymerase in 4: 1: a template polynucleotide that: (a) has a targetnucleotide sequence comprising at least one set of complementarynucleotide sequences, (b) forms a loop capable of base pairing when thecomplementary nucleotide sequence of (a) hybridizes, (c) forms a loop bythe annealing of its 3′-end to itself, and (d) whose 3′-end annealed toitself can be a starting point for complementary strand synthesis usingitself as template; 2: at least two types of primers providing staffingpoints for complementary strand synthesis at different locations on thetemplate polynucleotide loop; 3: at least one type of primer providing astaffing point for complementary strand synthesis at a locationdifferent from the primers of 2 in a loop formed by the templatepolynucleotide and/or an elongation product produced by the annealing ofthe primers of 2 to the template polynucleotide; 4: a DNA polymerasecatalyzing complementary strand synthesis accompanying stranddisplacement; and, 5: a substrate for complementary strand synthesis. 2.The method of claim 1, wherein said template polynucleotide has on its5′-end a nucleotide sequence complementary to an arbitrary region of itsown nucleotide sequence.
 3. The method of claim 2, wherein said templatepolynucleotide is produced by the following steps of: a) annealing afirst primer to a target nucleotide sequence, and conducting acomplementary strand synthesis reaction using this as a starting point,wherein said first primer (i) can provide at the 3′-end a starting pointfor complementary strand synthesis to a region that defines the 3′-sideof one of the strands that compose the target nucleotide sequence, and(ii) has on its 5′-side a nucleotide sequence complementary to anarbitrary region of a complementary strand synthesis reaction productthat uses this primer as a starting point; b) placing, in a conditionthat allows base pairing, the region to where a second primer is toanneal in the elongation product of the first primer synthesized in stepa), wherein said second primer (i) has on its 3′-end a nucleotidesequence providing a staffing point for complementary strand synthesisto a region that defines the 3′-side of a target nucleotide sequence inthe elongation product that uses the first primer as a staffing point,and (ii) has on its 5′-side a nucleotide sequence that is complementaryto an arbitrary region of a complementary strand synthesis reactionproduct that uses this primer as an starting point; c) annealing saidsecond primer to the region that can form base pairing in step b), andcarrying out complementary strand synthesis using this as a startingpoint; and, d) annealing the 3′-end of the elongation product of thesecond primer synthesized in step c) to itself, and carrying outcomplementary strand synthesis using itself as template.
 4. The methodof claim 3, wherein the two types of primers are a first primer and asecond primer, and at least one type of the primers is a loop primerproviding, between a region derived from each primer in the elongationproduct of the first primer or second primer and the arbitrary regionwith respect to each primer, a starting point for complementary strandsynthesis.
 5. The method of claim 4, wherein said loop primer is (i) afirst loop primer providing, between a region derived from the firstprimer in the elongation product of the first primer and the arbitraryregion with respect to the first primer, a starting point forcomplementary strand synthesis, and (ii) a second loop primer providing,between a region derived from the second primer in the elongationproduct of the second primer and the arbitrary region with respect tothe second primer, a staffing point for complementary strand synthesis.6. The method of claim 4, wherein said loop primer further comprises onits 5′-end a nucleotide sequence complementary to the arbitrary region.7. The method of claim 3, wherein each product of the first primer orsecond primer is converted to a single strand by displacing theelongation product of the first primer and/or second primer according tocomplementary strand synthesis from an outer primer that provides astarting point for complementary strand synthesis to the 3′-side of atemplate with respect to the first primer or second primer in step b)and/or step c).
 8. The method of claim 3, wherein the target nucleotidesequence is present as a double-stranded polynucleotide in step a), andthe region to which the first primer is annealed is made to form basepair bonds according to a complementary strand synthesis reaction usingthe arbitrary primer as a starting point.
 9. The method of claim 8,wherein step a) is carried out in the presence of a melting temperatureregulator.
 10. The method of claim 9, wherein the melting temperatureregulator is at least one compound selected from the group consisting ofbetaine, proline, dimethylsulfoxide, and trimethylamine N-oxide.
 11. Amethod for amplifying a template polynucleotide, comprising the step ofrepeating complementary strand synthesis using the templatepolynucleotide as template according to the method of claim 1, and alsocarrying out another polynucleotide synthesis reaction according to themethod of claim 1 using the elongation product resulting from thesynthesis reaction as a new template polynucleotide.
 12. A method fordetecting a target nucleotide sequence in a sample, comprising the stepof carrying out the amplification method of claim 11, and correlatingthe production of the amplification reaction product with the presenceof a target nucleotide sequence.
 13. The method of claim 12, wherein themethod of claim 11 is carried out in the presence of a polynucleotidedetecting agent, and whether or not an amplification reaction product isproduced is observed based on a signal change of the detecting agent.14. A method for detecting a mutation in a target nucleotide sequenceaccording to the detection method of claim 12, the method comprising thesteps of (i) blocking at least one of the complementary strand synthesisreactions selected from the complementary strand synthesis reactionscomposing the amplification method, when the target nucleotide sequenceis not the predicted nucleotide sequence, and (ii) observing theinhibition of the amplification reaction.
 15. The method of claim 14that uses the following first primer and second primer, wherein at leasteither the first primer or second primer comprises a checking sequenceon its 5′-side, wherein, a checking sequence refers to a nucleotidesequence in which (i) when a nucleotide sequence that composes aspecific region is not the predicted nucleotide sequence, a mismatchoccurs at the time when the 3′-end of the complementary strandsynthesized using the checking sequence as template anneals to thetarget nucleotide sequence, or its complementary strand, and (ii) acomplementary strand synthesis reaction that starts by using the 3′-endas a starting point is inhibited by this mismatch, the first primer (i)can provide at its 3′-end a starting point for complementary strandsynthesis to a region that defines the 3′-side of one of the strandsthat composes a target nucleotide sequence, and (ii) has on its 5′-sidea nucleotide sequence that is complementary to the arbitrary region ofthe complementary strand synthesis reaction product that uses thisprimer as a starting point, and the second primer (i) has a nucleotidesequence on its 3′-end that provides a staffing point for complementarystrand synthesis to a region that defines the 3′-side of a targetnucleotide sequence in an elongation product that uses the first primeras a starting point, and (ii) has on its 5′-side a nucleotide sequencethat is complementary to the arbitrary region of the complementarystrand synthesis reaction product that uses this primer as a staffingpoint.
 16. The method of claim 15, wherein when the nucleotide sequencethat composes the specific region is not the predicted nucleotidesequence, a mismatch occurs in the 2nd to 4th nucleotides from the3′-end of the complementary strand at the time when the complementarystrand synthesized by using a checking sequence as template anneals tothe target nucleotide sequence, or its complementary strand.
 17. Themethod of claim 15 that uses the following first loop primer and/orsecond loop primer as loop primers; provided that, when the loop primercomprises on its 5′-side a nucleotide that is complementary to thearbitrary region arranged on the 5′-side of the primer, or when thenucleotide sequence arranged on the 5′-side of the primer is a checkingsequence, a sequence in which the nucleotide for providing the mismatchin the checking sequence differs from the checking sequence is arrangedon the 5′-side of the loop primer: first loop primer: provides, betweena region derived from a first primer in an elongation product of thefirst primer and the arbitrary region with respect to the first primer,a staffing point for complementary strand synthesis; second loop primer:provides, between a region derived from a second primer in an elongationproduct of the second primer and the arbitrary region with respect tothe second primer, a staffing point for complementary strand synthesis.18. The method of claim 17, wherein, when the nucleotide sequence thatcomposes the specific region is not the predicted nucleotide sequence, amismatch occurs in the 2nd to 4th nucleotides from the 3′-end of thecomplementary strand at the time when the complementary strandsynthesized using a checking sequence as a template anneals to thetarget nucleotide sequence, or its complementary strand, and wherein,the nucleotide sequence arranged on the 5′-side of the first loop primerand/or second loop primer differs in the nucleotide that causes themismatch in the checking sequence.